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The 16th International Multiple Endocrine Neoplasia Workshop (MEN2019) held in Houston, TX, USA, focused on emerging topics in the pathogenesis and therapy of malignant endocrine tumors associated with MEN syndromes. With MEN-2 syndromes, the most common malignancy is medullary thyroid carcinoma (MTC). In the spirit of the original MEN meeting workshop model, the conference included didactic lectures and interactive working groups of clinicians and researchers focused on the state of science in MTC and ongoing challenges or unmet needs in the understanding of MTC and to develop strategies to address these issues.

Background: Thyroid cancer patients with radioiodine-refractory (RAI-R) disease, resulting from insufficient RAI delivery and/or RAI resistance, have increased mortality and limited treatment options. To date, studies have largely focused on tumor mutations associated with different stages of disease, which could provide prognostic value for RAI-R disease. It was hypothesized that germline variants contributing to intrinsic differences in iodine metabolism, tumor microenvironment, and/or immune surveillance are associated with RAI-R disease. Methods: Whole-genome genotyping data analysis was performed on 1145 Caucasian (CAU) patients, 244 of whom were RAI-R, and 55 African American (AA) patients, nine of whom were RAI-R. Germline-variant association studies were conducted using candidate genes involved in iodine metabolism or DNA-damage repair, as well as genome-wide association analysis. Initial data indicated several notable variants in a small number of patients (n = 7), who were later determined to be AA patients of >80% African ancestry (n = 37). This led to the study focusing on germline single nucleotide polymorphisms uniquely associated with RAI-R AA patients. Sanger sequencing was performed to validate risk alleles and identify the incidence of the common somatic mutations BRAF(V600E), NRAS(Q61R), and HRAS(Q61R) in AA patients whose primary tumor samples were available (28/55). Results: TG, BRCA1, and NSMCE2 haplotypes were identified as being uniquely associated with RAI-R AA patients of >80% African ancestry. All patients with the TG haplotype (n = 4) had a biochemical incomplete response to RAI therapy. Patients with the NSMCE2 haplotype (n = 4) were diagnosed at a young age (13, 17, 17, and 26 years old) with distant metastatic disease at initial diagnosis. The BRCA1 haplotype co-occurred in three out of four patients with the NSMCE2 haplotype. The incidence of BRAF(V600E) appears lower in papillary thyroid carcinomas from AA patients of >80% African ancestry (3/14; 21%) than in AA patients of

Background: Anaplastic thyroid carcinoma (ATC) is an aggressive thyroid cancer that requires a rapid diagnosis and treatment to achieve disease control. Gene mutation profiling of circulating cell-free DNA (cfDNA) in peripheral blood may help to facilitate early diagnosis and treatment selection. The relatively rapid turnaround time compared with conventional tumor mutation testing is a major advantage. The objectives of this study were to examine the concordance of ATC-related mutations detected in cfDNA with those detected in the corresponding tumor tissue, and to determine the prognostic significance of cfDNA mutations in ATC patients. Methods: The ATC patients who were diagnosed and treated at The University of Texas MD Anderson Cancer Center between January 2015 and February 2018 and who had cfDNA testing were included in this study. cfDNA was collected by blood draw and was analyzed by next-generation sequencing (NGS) using the Guardant360-73 gene platform. Results: A total of 87 patients were included in the study. The most frequently mutated genes detected in cfDNA were TP53, BRAF, and PIK3CA. In 28 treatment naive ATC patients, the concordance rate of detected mutations in TP53, BRAF(V600E), and PIK3CA between cfDNA and matched tissue NGS was 82.1%, 92.9%, and 92.9%, respectively. Patients with a PIK3CA mutation detected on cfDNA had worse overall survival (OS) (p = 0.03). This association was observed across various treatment modalities, including surgery, cytotoxic chemotherapy, radiation, and BRAF inhibitor (BRAFi) therapy. With regard to treatment, BRAFi therapy significantly improved ATC OS (p = 0.003). Conclusions: cfDNA is a valuable tool to evaluate a tumor's molecular profile in ATC patients. We identified high concordance rates between the gene mutations identified via cfDNA analysis and those identified from the NGS of the corresponding tumor tissue sequencing. Identified mutations in cfDNA can potentially provide timely information to guide treatment selection and evaluate the prognosis in patients with ATC.

Forty years ago, physicians caring for the J-kindred, a 100+ member family with multiple endocrine neoplasia type 2A (MEN2A), hypothesized that early thyroidectomy based on measurement of the biomarker calcitonin could cure patients at risk for development of medullary thyroid carcinoma (MTC). We re-evaluated 22 family members with proven RET proto-oncogene mutations (C634G) who underwent thyroidectomy and central lymphadenectomy between 1972 and 1994 based on stimulated calcitonin abnormalities. Current disease status was evaluated by serum calcitonin measurement and neck ultrasound in 18 of the 22 prospectively screened patients. The median age of the cohort at thyroidectomy was 16.5 years (range 9–24). The median duration of follow-up at the time of examination was 40 years (range 21–43) with a median current age of 52 years (range 34–65). Fifteen of the 18 patients had no detectable serum calcitonin (

Context Interpretation of calcitonin measurement to predict the prognosis of medullary thyroid carcinoma (MTC) requires multiple measurements over an extended time period, making it an imperfect biomarker for evaluating prognosis or disease behavior. Single circulating cell-free DNA (cfDNA) values have been shown to be a valuable prognostic marker for several solid tumors. Objective We tested the hypothesis that cfDNA containing the RET M918T mutation could be detected in the blood of patients with advanced MTC whose tumor harbored an M918T mutation and would be able to predict overall survival more reliably than calcitonin. Design The level of cfDNA containing RET M918T mutation was measured in the plasma of patients with MTC via droplet digital polymerase chain reaction. Patients Patients had a confirmed sporadic MTC diagnosis, a serum calcitonin measurement >100 pg/mL, and tumor tissue biopsy results providing RET M918T mutation status. There were 75 patients included in this study, 50 of whom harbored an RET M918T mutation by tissue biopsy. Results RET M918T cfDNA was detected in 16 of 50 patients (32%) with a positive tissue biopsy. The detection of RET M918T cfDNA strongly correlated with worse overall survival and more accurately predicted a worse outcome than calcitonin doubling time. Conclusions Liquid biopsy is able to detect RET M918T mutations in patient plasma with high specificity but low sensitivity. In patients with established somatic RET M918T mutations, the allelic fraction of circulating tumor DNA is prognostic for overall survival and may play a role in monitoring response to treatment.

Anaplastic thyroid carcinoma (ATC) is an aggressive disease that requires rapid diagnosis and multimodality treatment. Recent advances in targeted therapeutics have provided ATC patients with previously unavailable treatment options, which may improve clinical outcomes in the coming years. Continued development of high-throughput next-generation sequencing provides clinicians with an unparalleled ability to characterize the genomic background of tumors in order to guide treatment selection and clinical trial enrollment.Twenty-three patients with ATC treated at the University of Texas MD Anderson Cancer Center between August 2015 and April 2016 were evaluated. All patients underwent next-generation sequencing using an institutional tissue-based DNA platform (50 genes) and a commercially available cell-free circulating DNA (cfDNA) platform (70 genes).Sequencing data were successfully obtained for both platforms on all patients. The most commonly mutated genes noted on both platforms were TP53 (15/23; 65%) and BRAF (11/23; 48%). Concordance between the tumor and cfDNA data was high for BRAF, PIK3CA, NRAS, and PTEN and moderate for TP53. Concordance was highest in patients who underwent dual-platform sequencing prior to initiation of definitive treatment, and lowest in patients who underwent cfDNA analysis following treatment. Nineteen patients had treatment at the University of Texas MD Anderson Cancer Center following cfDNA sequencing. One patient was observed, and three patients opted for hospice. At the time of last contact, 15/23 (65%) patients were alive.Next-generation sequencing platforms offer clinicians an opportunity to identify targetable oncogenic events in ATC. To the authors' knowledge, this is the largest sequential cohort of ATC patients who have undergone targeted genomic profiling. Based on these data, utilization of both tumor-based and cfDNA analysis in the context of clinical-trial development and application is recommended. Integration of these or similar platforms in clinical-trial implementation may have the potential to transform clinical outcomes for patients with ATC.

Context Germline RET K666N mutation has been described as a pathogenic mutation with low disease penetrance for medullary thyroid cancer (MTC) without other features of multiple endocrine neoplasia type 2A. We describe a patient with homozygous RET K666N mutation with MTC and bilateral pheochromocytoma (PHEO). Case Description A 59-year-old woman received a diagnosis of MTC after biopsy of two thyroid nodules. Coincident biochemical and radiologic testing was suspicious for bilateral PHEO, confirmed after bilateral adrenalectomy. There was no evidence of primary hyperparathyroidism (PHPT). She had a total thyroidectomy with neck dissection revealing bilateral MTC with lymph node metastases. Germline RET testing identified homozygous K666N mutations. Genetic testing of family members showed that both adult children harbor a heterozygous K666N mutation. Her 32-year-old son had an elevated calcitonin level and underwent thyroidectomy, which identified MTC. Her 30-year-old daughter had a normal calcitonin level. Prophylactic thyroidectomy showed C-cell hyperplasia only. Three of seven other family members were tested and found to carry the mutation. All had normal calcitonin levels, and none had biochemical evidence of PHEO or PHPT. Given the absence of PHEO in reported RET K666N families, our proband underwent genetic testing for causes of hereditary paragangliomas or PHEO. No additional mutations were identified. Conclusions Here we report a case of a homozygous RET K666N mutation leading to coincident MTC and PHEO. Heterozygous presentations of RET K666N mutations have low penetrance for isolated MTC. We believe that the gene dosage associated with the homozygosity of this variant contributed to the occurrence of bilateral PHEO.

Oncogenic RET tyrosine kinase gene fusions and activating mutations have recently been identified in lung cancers, prompting initiation of targeted therapy trials in this disease. Although RET point mutation has been identified as a driver of tumorigenesis in medullary thyroid carcinoma (MTC), no fusions have been described to date.We evaluated the role of RET fusion as an oncogenic driver in MTC.We describe a patient who died from aggressive sporadic MTC10 months after diagnosis. Her tumor was evaluated by means of next-generation sequencing, including an intronic capture strategy.A reciprocal translocation involving RET intron 12 was identified. The fusion was validated using a targeted break apart fluorescence in situ hybridization probe, and RNA sequencing confirmed the existence of an in-frame fusion transcript joining MYH13 exon 35 with RET exon 12. Ectopic expression of fusion product in a murine Ba/F3 cell reporter model established strong oncogenicity. Three tyrosine kinase inhibitors currently used to treat MTC in clinical practice blocked tumorigenic cell growth.This finding represents the report of a novel RET fusion, the first of its kind described in MTC. The finding of this potential novel oncogenic mechanism has clear implications for sporadic MTC, which in the majority of cases has no driver mutation identified. The presence of a RET fusion also provides a plausible target for RET tyrosine kinase inhibitor therapies.

Context High-risk RET mutations (codon 634) are associated with earlier development of medullary thyroid carcinoma (MTC) and presumed increased aggressiveness compared with moderate-risk RET mutations. Objective To determine whether high-risk RET mutations are more aggressive. Design Retrospective cohort study using institutional multiple endocrine neoplasia type 2 registry. Setting Tertiary cancer care center. Patients Patients with MTC and moderate- or high-risk germline RET mutation. Intervention None (observational study). Main Outcome Measures Proxies for aggressiveness were overall survival (OS) and time to distant metastatic disease (DMD). Results A total of 127 moderate-risk and 135 high-risk patients were included (n = 262). Median age at diagnosis was 42.3 years (range, 6.4 to 86.4 years; mean, 41.6 years) for moderate-risk mutations and 23.0 years (range, 3.7 to 66.8 years; mean, 25.6 years) for high-risk mutations (P < 0.0001). Moderate-risk patients had more T3/T4 tumors at diagnosis (P = 0.03), but there was no significant difference for N or M stage and no significant difference in OS (P = 0.40). From multivariable analysis for OS, increasing age [hazard ratio (HR), 1.05/y; 95% confidence interval (CI), 1.03 to 1.08], T3/T4 tumor (HR, 2.73; 95% CI, 1.22 to 6.11), and M1 status at diagnosis (HR, 3.93; 95% CI, 1.61 to 9.59) were significantly associated with worse OS but high-risk mutation was not (P = 0.40). No significant difference was observed for development of DMD (P = 0.33). From multivariable analysis for DMD, only N1 status at diagnosis was significant (HR, 2.10; 95% CI, 1.03 to 4.27). Conclusions Patients with high- and moderate-risk RET mutations had similar OS and development of DMD after MTC diagnosis and therefore similarly aggressive clinical courses. High-risk connotes increased disease aggressiveness; thus, future guidelines should consider RET mutation classification by disease onset (early vs late) rather than by risk (high vs moderate).

Anaplastic thyroid cancer (ATC) is one of the most lethal forms of cancer with a high mortality rate. Current guidelines support surgery for resectable ATC followed by external beam radiation therapy (EBRT) with or without chemotherapy. Treatment for those who are unresectable is palliative. Our goal was to examine first-line therapies as well as the role of genomic profiling in an effort better understand how to approach ATC.This is a retrospective study of ATC patients who were seen at our institution from January 2013 to October 2015. Median overall survival (OS) and time to treatment failure (TTF) were calculated by the Kaplan-Meier method.Fifty-four patients were included. Median age at diagnosis was 63 years and 29/54 (54%) were women. The majority had stage IVC disease at diagnosis (50%), followed by IVB (32%), and IVA (18%). Approximately 93% had somatic gene testing. Initial treatment was surgery in 23 patients, EBRT with or without radiosensitizing chemotherapy in 29 patients, and systemic chemotherapy in 2 patients. Nineteen patients had all three treatment modalities. For the entire cohort, median OS was 11.9 months with 39% survival at 1 year and median TTF was 3.8 months. The majority of patients (74%) developed new distant metastasis or progression of existing metastatic disease. Patients who received trimodal therapy consisting of surgery, EBRT, and chemotherapy had a median OS of 22.1 months versus 6.5 months in those who received dual therapy with EBRT and chemotherapy (p = 0.0008). The TTF was the same in the two groups (7.0 and 6.5 months, respectively). Men were three times more likely to die from ATC than women (p = 0.0024). No differences in OS or TTF were noted based on tumor size (5 cm cutoff), age (60 years cutoff), or presence of any mutation. There was a trend toward shorter TTF in patients with somatic mutations in TP53.Patients with ATC amenable to aggressive tri-modal therapy demonstrate improved survival. The short TTF, due primarily to distant metastatic disease, highlights the potential opportunity for improved outcomes with earlier initiation of systemic therapy including adjuvant or neoadjuvant therapy.

Metastatic pheochromocytomas and paragangliomas (MPPs) present clinicians with three major challenges: scarcity, complexity of characterization, and heterogeneous behavior and prognosis. As with the treatment for all neuroendocrine tumors, the control of hormonal symptoms and tumor growth is the main therapeutic objective in MPP patients. A significant number of MPP patients still die from uncontrolled hormone secretion. In addition, the management of MPPs remains palliative. Steps forward include proper characterization of MPP patients at large cancer referral centers with multidisciplinary teams; improved strategies to stratify patients prognostically; and implementation of trials within national and international networks. Progress in the molecular characterization and staging of MPPs constitutes the basis for significant treatment breakthroughs.

Background: Age-related risk of medullary thyroid carcinoma (MTC) development in presymptomatic carriers of lower risk germline RET mutations is uncertain; such data may aid counseling patients regarding timing of thyroidectomy. Methods: From an institutional database and an exhaustive literature review, we identified 679 patients with American Thyroid Association (ATA) level A or B mutations who were identified because of family screening (index cases of MTC were excluded to minimize selection bias). We evaluated age at thyroidectomy or last evaluation if no thyroidectomy, preoperative calcitonin level (elevated or not), the mutated codon, and outcome (MTC vs. no MTC after thyroidectomy or no clinical evidence of MTC if thyroid intact). Data were used to estimate the cumulative prevalence of MTC and/or assess likelihood of MTC stratified by codon. After exclusion of cases with missing data or small representation, 503 patients with mutations in codons 533, 609, 611, 618, 620, 791, and 804 were analyzed. Results: 236 patients had MTC. Cumulative prevalence and median time to MTC varied by codon and within ATA risk levels (p

BackgroundMultiple endocrine neoplasia type 2 is an autosomal dominant inherited syndrome caused by activating mutations in the RET proto-oncogene. The RETK666N DNA variant was previously reported in two isolated medullary thyroid carcinoma (MTC) cases, but no family studies are available, and its oncogenic significance remains unknown.MethodsThe clinical features, genetic data, and family information of eight index MTC patients with a germline RETK666N variant were assessed.ResultsFour probands presented with MTC and extensive nodal metastasis, one with biopsy-confirmed distant metastasis. Two additional probands presented with localized disease. However, nodal status was not available. Of the final two probands, one had an incidental 1.5 mm MTC and C-cell hyperplasia uncovered after surgery for papillary thyroid carcinoma, and one had two foci of MTC (largest dimension 2.3 cm) detected after surgery for dysphagia. Genetic screening identified 16 additional family members carrying the K666N variant (aged 5-90 years), 11 of whom have documented evaluation for MTC. Of these, only two were found to have elevated basal serum calcitonin upon screening, and the remaining patients had calcitonin levels within the reference range. One patient who elected to have a thyroidectomy at 70 years of age was confirmed to have MTC. The other subject, 57 years old, elected surveillance. Four prophylactic thyroidectomies were performed, with one case of C-cell hyperplasia at 20 years and three cases that revealed normal pathology at ages 21, 30, and 30 years. None of the K666N DNA variant carriers had evidence of primary hyperparathyroidism or pheochromocytoma.ConclusionsFrom this case series, the largest such experience to date, it is concluded that the RETK666N variant is likely pathogenic and associated with low penetrance of MTC. However, the findings are insufficient to define its pathogenicity clearly and make firm recommendations for screening and treatment. Given the potential benefit associated with early detection of aberrant C-cell growth, and the noninvasive nature of genetic testing, "at risk" individuals should be screened, and if the K666N variant is identified, they should be managed using a personalized screening approach for detection of MTC.

Context: Individual patient prognostication for advanced thyroid cancer (TC) is challenging. Circulating tumor cells (CTCs) have been shown to be a valuable prognostic marker for other solid cancers. Objective: We hypothesized that CTCs are present in the blood of patients with advanced TC and their number can predict overall survival (OS). Setting: This is a prospective study at a tertiary cancer hospital. Patients, Interventions, and Main Outcome Measures: Initial studies were performed with TC cell lines to determine the feasibility of detection using the Veridex CellSearch. CTC enumeration was performed in blood samples from 18 patients with distantly metastatic medullary TC (metMTC), 14 with distantly metastatic differentiated TC (metDTC), and 10 controls with a history of TC but no evidence of disease. The prognostic value of CTC levels to predict OS in metMTC patients was assessed. Results: CellSearch detected cells from MTC and DTC but not anaplastic TC cell lines. Six metMTC patients but no metDTC or control patients had more than or equal to 5 CTCs detected by the CellSearch assay. Median survival in metMTC patients with more than or equal to 5 CTCs was 13 months vs 51.5 months for those with less than 5 CTCs (P = .0116). The hazard ratio for mortality of patients with more than or equal to 5 CTCs compared with those with less than 5 CTCs was 3.95 (1.20–13.0, P = .0245). Conclusions: The presence of more than or equal to 5 CTCs in patients with metMTC is associated with worse OS. Larger cohorts are required to validate the prognostic value of CTC enumeration.

Background: The cyclin-dependent-kinase inhibitors (CDKN)/retinoblastoma (RB1) pathway has been implicated as having a role in medullary thyroid carcinoma (MTC) tumorigenesis. CDKN2C loss has been associated with RET-mediated MTC in humans but with minimal phenotypic correlation provided. The objective of this study was to evaluate the association between tumor RET mutation status, CDKN2C loss, and aggressiveness of MTC in a cohort of patients with sporadic disease. Methods: Tumors from patients with sporadic MTC treated at a single institution were evaluated for somatic RET(M918T) mutation and CDKN2C copy number loss. These variables were compared to patient demographics, pathology detail, clinical course, and disease-specific and overall survival. Results: Sixty-two MTC cases with an initial surgery date ranging from 1983 to 2009 met the inclusion criteria, of whom 36 (58%) were male. The median age at initial surgery was 53 years (range 22–81 years). The median tumor size was 30 mm (range 6–145 mm) with 29 (57%) possessing extrathyroidal extension. Nodal and/or distant metastasis at presentation was found in 47/60 (78%) and 12/61 (20%) patients, respectively. Median follow-up time was 10.5 years (range 1.1–27.8 years) for the censored observations. The presence of CDKN2C loss was associated with worse M stage and overall AJCC stage. Median overall survival of patients with versus without CDKN2C loss was 4.14 [confidence interval (CI) 1.93–NA] versus 18.27 [CI 17.24–NA] years (p

Pheochromocytoma (PHEO) occurs in 50% of patients with multiple endocrine neoplasia type 2 (MEN2). It is unknown if the presence of PHEO is associated with more aggressive medullary thyroid cancer (MTC).To present our experience with MEN2 PHEO and evaluate whether PHEO impacts MTC overall survival in patients with RET codon 634 mutations.We performed a retrospective chart review of MEN2 patients at MD Anderson Cancer Center from 1960 through 2012.The study group comprised 85 patients (group 1) with MEN2-associated PHEO. Of these, 59 patients (subgroup 1) with RET codon 634 mutations were compared to 48 patients (group 2) with RET codon 634 mutations, but without MEN2-associated PHEO.Of 85 patients with MEN2 and PHEO, 70 had MEN2A and 15 had MEN2B. Median age at PHEO diagnosis was 32 years. The initial manifestation of MEN2 was MTC in 60% of patients, synchronous MTC and PHEO in 34%, and PHEO in 6% of patients. Of patients, 72% had bilateral PHEO, and most tumors were synchronous (82%). Subgroup analysis of MEN2 patients with and without PHEO, who were carriers of RET codon 634, the most common mutation with PHEO, showed no significant differences in the stage of MTC at initial diagnosis. The median follow-up time for patients with PHEO was 249 months and without PHEO was 67 months (P.01). Survival analyses among RET 634 carriers did not show shorter survival for patients with PHEO. The median survival time for patients with PHEO was 499 months and without PHEO was 444 months (P.05).PHEO in MEN2 patients are usually bilateral and unlikely to be metastatic. Subgroup analysis of patients with RET 634 mutations with and without PHEO showed that PHEO was not associated with a more advanced stage of MTC at diagnosis or a shorter survival.

Medullary thyroid carcinoma (MTC) and the multiple endocrine neoplasia (MEN) type 2 syndromes are rare but important endocrine diseases that are increasingly managed by pediatric providers. MTC is generally associated with a favorable prognosis when diagnosed during childhood, where it frequently occurs secondary to activating mutations in the RET proto-oncogene and arises from pre-existing C-cell hyperplasia. MEN2A accounts for 90-95% of childhood MTC cases and is most commonly due to mutations in codon 634 of RET. MEN2B is associated with the most aggressive clinical presentation of MTC and is almost always due to the Met918Thr mutation of RET. Surgery is the primary treatment and only chance of cure, although the advent of targeted therapies seems to be improving progression-free survival in advanced cases. Since the discovery of the role of RET in MEN2A, considerable advances in the management of this syndrome have occurred, and most of the children with MEN2A who have undergone early thyroidectomy will now lead full, productive lives. Strong genotype-phenotype correlations have facilitated the development of guidelines for interventions. Contemporary approaches for deciding the appropriate age at which surgery should take place incorporate data from ultrasonography and calcitonin measurements in addition to the results of genotyping. To optimize care and to facilitate ongoing research, children with MTC and the MEN2 syndromes are optimally treated at tertiary centers with multidisciplinary expertise.

Objective To evaluate the expression of various phosphaturic factors in the tumor of a patient with tumorinduced osteomalacia (TIO) and to analyze serum levels of fibroblast growth factor (FGF)-23 in TIO and healthy subjects. Methods We measured serum FGF-23 levels in 2 patients with TIO and 6 healthy volunteers. Expression of FGF-23, matrix extracellular phosphoglycoprotein (MEPE), FGF-7, and secreted frizzled-related protein-4 (sFRP-4) was analyzed in a hemangiopericytoma from a patient with TIO, in a hemangiopericytoma from a patient without TIO, and in various control cell lines. Results Serum FGF-23 levels were substantially higher in patients with TIO in comparison with those in healthy control subjects and normalized with successful resection of the tumor. Tissue expression analysis showed preferential expression of FGF-23 and MEPE in TIO related hemangiopericytoma, whereas FGF-7 and sFRP-4 were widely expressed in all studied cell lines and tissues. Conclusion These results support the use of FGF-23 measurements for the diagnosis and follow-up of patients with TIO. In addition, the specific expression of FGF-23 and MEPE in the TIO-associated tumor suggests an important role of these two phosphatonins in the pathogenesis of TIO. Because of the limited number of patients in our report, further studies are needed to clarify the role of different phosphatonins in the development of the clinical features of TIO. (Endocr Pract. 2008;14:1108-1114)

Activating mutations in the RET protooncogene have been demonstrated in multiple endocrine neoplasia 2 and sporadic medullary thyroid carcinoma (MTC). However, the complete genetic etiology underlying MTC tumorigenesis remains unclear.Our objective was to define more precisely the chromosomal regions and uncover novel genes associated with MTC tumorigenesis.In this study, we used high resolution array-based comparative genomic hybridization to define tumor-associated copy number alterations (CNA) in 30 primary MTCs: 20 sporadic tumors (50% of which harbored RET mutation), and 10 hereditary.We identified 98 CNA, including 76 genomic allelic losses, two gains, and 20 copy number variations associated with MTC. Across sporadic and hereditary groups, there was a similar and overlapping pattern of predominant allelic loss. There were 29 regions containing at least 30% CNA in the 30 tumor samples. The most frequent allelic loss occurred in four loci, 7q36.1, 12p13.31, 13q12.11, and 19p13.3-11. No regions were found to be uniquely altered in the hereditary tumors. There were 21 CNA specific to sporadic MTC, with loss of 11q23.3 uniquely altered in RET negative tumors. Pathway analysis found cellular growth and proliferation as the most significant overall target, and cell death as the most significant pathway targeted in sporadic MTC.Our findings underscore the importance of candidate tumor suppressor genes together with RET alterations in MTCs. Despite of RET status, all MTC might share similar oncogenetic mechanisms. Dysfunction of cell proliferation and cell death may both be involved in MTC tumorigenesis.

Adrenocortical carcinoma is a rare malignancy with poor prognosis and limited response to chemotherapy. Hepatocyte growth factor (HGF) and its receptor cMET augment cancer growth and resistance to chemotherapy, but their role in adrenocortical carcinoma has not been examined. In this study, we investigated the association between HGF/cMET expression and cancer hallmarks of adrenocortical carcinoma. Transcriptomic and immunohistochemical analyses indicated that increased HGF/cMET expression in human adrenocortical carcinoma samples was positively associated with cancer-related biologic processes, including proliferation and angiogenesis, and negatively correlated with apoptosis. Accordingly, treatment of adrenocortical carcinoma cells with exogenous HGF resulted in increased cell proliferation in vitro and in vivo while short hairpin RNA–mediated knockdown or pharmacologic inhibition of cMET suppressed cell proliferation and tumor growth. Moreover, exposure of cells to mitotane, cisplatin, or radiation rapidly induced pro-cMET expression and was associated with an enrichment of genes (e.g., CYP450 family) related to therapy resistance, further implicating cMET in the anticancer drug response. Together, these data suggest an important role for HGF/cMET signaling in adrenocortical carcinoma growth and resistance to commonly used treatments. Targeting cMET, alone or in combination with other drugs, could provide a breakthrough in the management of this aggressive cancer. Cancer Res; 75(19); 4131–42. ©2015 AACR.

Sorafenib, a tyrosine kinase inhibitor, is a common first-line therapy for advanced differentiated thyroid cancer (DTC). However, responses are not durable and drug toxicity remains a problem.The objective of the study was to determine the efficacy of salvage therapy after first-line sorafenib failure.This was a retrospective review at M. D. Anderson Cancer Center from January 2005 to May 2013.The study included patients with metastatic DTC who received salvage therapy after their initial sorafenib failure (group 2). PATIENTS who received first-line sorafenib only (group 1) were evaluated for comparison of overall survival (OS).Progression-free survival, best response, and median OS were measured.Sixty-four patients with metastatic, radioactive iodine refractory DTC were included; 35 were in group 1 and 25 were in group 2, and the groups were well balanced. Median OS of all 64 patients receiving first line sorafenib was 37 months; median OS was significantly longer with salvage therapy compared with sorafenib alone (58 vs 28 months, P = .013). In group 2, 17 patients were evaluable for best response, although two patients had toxicity with sorafenib, which was discontinued before restaging. Best responses with first-line sorafenib were partial response in 2 of 15 (13%), stable disease in 10 of 15 (67%), and progressive disease in 3 of 15 (20%) patients. With salvage therapy, partial responses were seen in 7 of 17 (41%) and stable disease in 10 of 17 (59%) patients. Median progression-free survival was 7.4 months with first-line sorafenib and 11.4 months with salvage therapy. Salvage therapy included sunitinib (n = 4), pazopanib (n = 3), cabozantinib (n = 4), lenvatinib (n = 3), and vemurafenib (n = 3).Other targeted agents are effective salvage treatments after sorafenib failure, despite similar mechanisms of action, and should be offered to patients who are able to receive salvage therapy.

We report the cloning, genomic organization and sequence of the mouse alpha-CALC and beta-CALC genes. The two genes share extensive sequence homology. The transcription units of both genes contain 6 exons. Transcripts of the alpha-CALC gene were found to alternatively include exon 4 or exons 5 and 6. For the beta-CALC gene exon 4 was not detected in transcripts derived from this gene. The predicted mouse alpha-CGRP was found to be identical to rat alpha-CGRP, however, beta-CGRP predicted amino acid sequences revealed three amino acid differences suggesting these residues are not critical to CGRP function.

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that is widely expressed, has tissue-specific functions, and regulates cell growth. Activating mutations of this receptor cause autosomal dominant hypocalcemia, a syndrome characterized by hypocalcemia and hypercalciuria. The identification of a family with an activating mutation of the CaR (Thr151Met) in which hypocalcemia cosegregates with several unusual neoplasms led us to examine the transforming effects of this mutant receptor. Transfection of NIH/3T3 cells with the mutant but not the normal receptor supported colony formation in soft agar at subphysiologic calcium concentrations. The mutant CaR causes a calcium-dependent activation of the extracellular signal-regulated protein kinase (ERK) 1/2 and Jun-N-terminal kinase/stress-activated (JNK/ SAPK) pathways, but not P38 MAP kinase. These findings contribute to a growing body of information suggesting that this receptor plays a role in the regulation of cellular proliferation, and that aberrant activation of the mutant receptor in this family may play a role in the unusual neoplastic manifestations.

Mutations in genes encoding the ATP-regulated potassium (K(ATP)) channels of the pancreatic beta-cell (SUR1 and Kir6.2) are the major known cause of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We collected all cases of PHHI diagnosed in Finland between 1983 and 1997 (n = 24). The overall incidence was 1:40,400, but in one area of Central Finland it was as high as 1:3,200. Haplotype analysis using polymorphic markers spanning the SUR1/Kir6.2 gene cluster confirmed linkage to the 11p region. Sequence analysis revealed a novel point mutation in exon 4 of SUR1, predicting a valine to aspartic acid change at amino acid 187 (V187D). Of the total cases, 15 affected individuals harbored this mutation in heterozygous or homozygous form, and all of these had severe hyperinsulinemia that responded poorly to medical treatment and required subtotal pancreatectomy. No K(ATP) channel activity was observed in beta-cells isolated from a homozygous patient or after coexpression of recombinant Kir6.2 and SUR1 carrying the V187D mutation. Thus, the mutation produces a nonfunctional channel and, thereby, continuous insulin secretion. This unique SUR1 mutation explains the majority of PHHI cases in Finland and is strongly associated with a severe form of the disease. These findings provide diagnostic and prognostic utility for suspected PHHI patients.

Application of RET proto-oncogene mutation analysis to the clinical management of MEN 2 and FMTC has simplified and enhanced the power of earlier used screening and treatment efforts for hereditary MTC. The approaches outlined herein are cost-effective, have improved diagnostic accuracy, and hold the promise of improved cure rates for this neoplasm. Further studies to elucidate the mechanism by which these activating mutations cause transformation may lead to other strategies for prevention or treatment of this neoplasm.

The identification of RET proto-oncogene mutations in patients with MEN2 2 years ago was a watershed event in the management of this genetic cancer syndrome. The identification of a finite number of mutations that together causes more than 95% of hereditary and 15-25% of sporadic MTC has made it possible to develop simple and definitive tests to screen individuals at risk for this tumour syndrome. The impact of this technology is enormous. It is now possible to reassure 50% of family members at risk that they, and their children, do not have to worry about developing MTC. In the other 50% who are gene carriers, it is now possible to approach clinical management with greater certainty and plot strategies that are likely to result in a greater percentage of curative therapy. It seems likely that this technology will also have an impact on the management of sporadic MTC, although it is still too early to define a specific role for mutational analysis in these patients, except to exclude hereditary disease. The identification of specific mutations causative for MTC makes it possible to conceive future strategies for the treatment or prevention of MTC and to further extend the impact of these exciting findings.

The primary transcript of the calcitonin (CT)/calcitonin gene-related peptide (CGRP) is alternatively spliced in a cell-specific fashion to produce CT in thyroid C cells and CGRP in neuronal cells. The key step in this regulatory process is the recognition and inclusion of exon 4 to produce CT mRNA or nonrecognition and exclusion of exon 4 to produce CGRP mRNA. To determine whether inclusion/exclusion of CT exon is regulated independently of its position, we created a series of minigene constructs containing decreasing amounts of CT gene sequence. A human glioblastoma cell line, T98G, was tested and used as a CT exon exclusion cell line, while HeLa cells were used as a CT exon inclusion cell line. CT exon inclusion/exclusion was regulated when either the relative position of exon 4 within the CT gene was changed or when the exon with flanking sequence was inserted into a completely heterologous gene. Our results demonstrate that CT exon functions as a unit in a position-independent fashion in regulating its own inclusion/exclusion. We believe that the heterologous fusion gene containing only exon 4 and part of its flanking intron sequences will be useful for further defining the sequence elements involved in the regulation of CT/CGRP splicing.

Most cases of multiple endocrine neoplasia type 2B (MEN-2B) are attributable to a germline methionine to threonine mutation at codon 918 (M918T) of the RET proto-oncogene; very few cases of a germline alanine to phenylalanine mutation at codon 883 (A883F) are reported without a clear description of the clinical course. Nevertheless, RET-A883F is currently considered to be among the highest risk mutations, and prophylactic thyroidectomy is recommended as early as 6 months of life. Further characterization of the clinical behavior of RET-A883F mutation is warranted. We present the clinical data for a family with MEN-2B associated with RET-A883F mutation.The proband, a 39-year-old woman, had multifocal medullary thyroid carcinoma (MTC) with cervical lymphadenopathy, but no evidence of distant metastases. She was disease free after surgical resection. She also had bilateral pheochromocytomas and mucosal neuromas leading to the clinical diagnosis of MEN-2B. Genetic testing showed that the woman and her three children (3-5 years old) had the RET-A883F mutation. The children had near-normal calcitonin levels, and none had sonographic evidence of suspicious thyroid nodules or cervical lymphadenopathy.A family with MEN-2B due to RET-A883F mutation displayed a less aggressive form of MTC than what is usually seen in patients with RET-M918T mutation. RET-A883F mutation could be a lower-risk mutation than previously thought and the current recommendation of prophylactic thyroidectomy in the first year of life may not be warranted. Further reports will help clarify the natural history of MTC caused by this mutation.

Pheochromocytomas and sympathetic paragangliomas are rare neuroendocrine tumors for which no precise histological or molecular markers have been identified to differentiate benign from malignant tumors.The aim was to determine whether primary tumor location and size are associated with malignancy and decreased survival.We performed a retrospective chart review of patients with either pheochromocytoma or sympathetic paraganglioma.The study group comprised 371 patients.Overall survival and disease-specific survival were analyzed according to tumor size and location.Sixty percent of patients with sympathetic paragangliomas and 25% of patients with pheochromocytomas had metastatic disease. Metastasis was more commonly associated with primary tumors located in the mediastinum (69%) and the infradiaphragmatic paraaortic area, including the organ of Zuckerkandl (66%). The primary tumor was larger in patients with metastases than in patients without metastatic disease (P0.0001). Patients with sympathetic paragangliomas had a shorter overall survival than patients with pheochromocytomas (P0.0001); increased tumor size was associated with shorter overall survival (P0.001). Patients with sympathetic paragangliomas were twice as likely to die of disease than patients with pheochromocytomas (hazard ratio = 1.93; 95% confidence interval = 1.20-3.12; P = 0.007). As per multivariate analysis, the location of the primary tumor was a stronger predictor of metastases than was the size of the primary tumor.The size and location of the primary tumor were significant clinical risk factors for metastasis and decreased overall survival duration. These findings delineate the follow-up and treatment for these tumors.

Sympathetic paragangliomas are autonomic nervous system tumors associated with dysregulation of intracellular oxygen metabolism. Exposure to high altitudes is reported to activate the production of catecholamines in the sympathoadrenal system. We describe an individual with a paraganglioma complicated by a catecholamine crisis that occurred on the summit of Mount Kilimanjaro.

Medullary thyroid carcinoma (MTC) is a cancer of the parafollicular C cells commonly caused by an inherited or acquired RET proto-oncogene mutation. Therapeutic resistance and recurrence of the disease imply the presence of cancer stem cells in MTC.In this study, we sought to identify and characterize cancer stem cell-like cells in MTC.The characterization of stem cell properties was performed using immunostaining, flow cytometry, sphere formation assay, rederivation assay, Western blotting, and quantitative RT-PCR of defined markers of neural stem and progenitor cells. The role of ret proto-oncogene activation was assessed through RNA interference knockdown.CD133 positivity was identified by immunostaining patient MTC. Flow cytometry confirmed a subpopulation of CD133(+) cells in two MTC cell lines. The CD133(+) cells could be expanded by sphere formation assay, passaged multiple times, and expressed neural progenitor markers beta-tubulin 3 and glial fibrillary acidic protein. The MZ-CRC-1 cell line, which harbors a M918T RET mutation, had greater CD133(+) cell numbers and sphere-forming ability than the TT cell line, which harbors the less active C634W mutation. Sphere formation was more dependent on ret proto-oncogene activity than epidermal growth factor or fibroblast growth factor.Our data support the existence of cancer stem-like cells in MTC, which exhibit the features of self-renewal and of multiple lineage differentiation that is dependent on ret proto-oncogene receptor activity. These findings may provide new insights to develop more promising therapy for MTC.

The pre-mRNA encoding calcitonin (CT) and CT gene-related peptide (CGRP) is differentially processed in a tissue-specific fashion to include exon 4 (which encodes CT) or exclude this exon and splice to exon 5 (which encodes CGRP). We have used a CT-specific in vitro RNA-processing system to identify cis-acting sequences required to prevent splicing to exon 5. Deletion mapping demonstrated the presence of an element within the first 45 nucleotides of the CT-specific exon 4 that was required to suppress splicing to the CGRP-specific exon 5. This element was able to function in a completely heterologous system to suppress splicing when the CGRP exon was replaced with a constitutive viral exon. The element was unable to suppress splicing in the absence of a proximal CT-specific 3' splice site. Our results suggest that CT-specific splicing requires assisted recognition of its 3' splice site.

The calcitonin (CT) gene is expressed normally in thyroidal C-cells and in a restricted population of cells in the central and peripheral nerve system. To define the cis-elements within the 5'-flanking DNA of the human CT gene which mediate this cell-specific expression, we used DNA transfer techniques and a transient transfection approach. We found that a DNA sequence located between -1290 and -820 of the CT 5'-flanking DNA functioned as an enhancer of basal transcription in C-cells (from medullary thyroid carcinoma) but not in rat glioma (C6), hamster insulinoma (HIT), fibroblasts (3T3), or epithelial cells (HeLa and CV1). Further mapping revealed the presence of at least two elements within the enhancer region; an upstream element (USE, located between -1060 and -1030) which could not function independently but its removal caused 70-80% loss of enhancer activity and a downstream element (DSE, located at -1033 to -920) which functioned independently as a cell-specific enhancer but with reduced activity. The binding pattern of nuclear proteins from C-cells to the enhancer elements was studied by an electrophoretic mobility shift assay. A protein-DNA complex was formed with the USE which could be competed, specifically, by an oligonucleotide containing the microE2 motif of the immunoglobulin gene enhancer. A similar complex was formed with the DSE fragment. Nuclear proteins from HeLa cells failed to form complexes with USE. Moreover, the binding pattern of proteins derived from HeLa cells to DSE was different from that of C-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaplastic thyroid carcinoma (ATC) can occur in the setting of differentiated thyroid carcinoma (DTC), which suggests a continuum in malignant progression from DTC to ATC. The Ras/Raf-MAPK and the phosphatidylinositol 3-kinase/Akt signaling pathways play critical roles in DTC tumorigenesis, but their roles in the pathogenesis of ATC are poorly defined.Our objective was to explore the potential contributions of these two pathways in ATC pathogenesis.The mutational status of BRAF, PIK3CA, PTEN, and RAS genes was analyzed in genomic DNA from microdissected tumor specimens of 36 cases of ATC, and in 16 samples of paired-matched lymph node metastases. PIK3CA copy number gain was assessed by real-time quantitative PCR. We performed immunohistochemistry for phospho-ERK and phospho-AKT in 26 cases of ATC.DTC was present in half of the cases. BRAF V600E mutation was identified in nine of 36 (25%) ATCs; seven cases had identical mutations in both the ATC and DTC components. PIK3CA kinase domain mutations were found in five (14%) ATCs, one of which had mutations in both differentiated and anaplastic areas. RAS and PTEN mutations were each found in two (6%) ATCs. PIK3CA gain copy number was found notably increased in 14 (39%) ATCs.BRAF mutations appear to play a role in the tumorigenesis of a subset of ATCs, and the majority of lymph node metastases. PIK3CA alterations occur preferentially in the later stages of ATC and were the most relevant events during thyroid cancer progression. The activation of both pathways suggests an important role in ATC dedifferentiation.

Context: The recent identification of germline mutations of the mitochondrial complex II genes in variants of paraganglioma/pheochromocytoma syndrome has enlarged the number of known causative genes for hereditary pheochromocytoma. A question confronting clinicians is whether they should screen patients with apparently sporadic pheochromocytomas for unsuspected germline mutations of some or all of the seven genes known to cause hereditary paraganglioma or pheochromocytoma (NF1, VHL, RET, MEN1, SDHD, SDHC, and SDHB). A positive answer was suggested by a report that placed the estimate of hereditary disease in apparently sporadic pheochromocytoma as high as 24%. Evidence Acquisition: We applied clinically useful criteria to a review of the literature, defining cases of apparently sporadic pheochromocytoma as those without a suspicious personal or family history, with a focal, unilateral pheochromocytoma, and presenting at age less than 50 yr. Evidence Synthesis: We reduced the overall estimate of unsuspected hereditary pheochromocytoma patients with apparently sporadic pheochromocytoma to approximately 17%. Mutations in only three genes (VHL, SDHB, and SDHD) accounted for almost this entire minority, and unsuspected RET mutation was rare. Costs, coverage by insurance, the potential effect on insurability, and deficient information for populations outside of referral centers should be considered before recommending genetic testing in patients with apparently sporadic presentations of pheochromocytomas. Conclusion: We recommend genetic testing for patients with an apparently sporadic pheochromocytoma under the age of 20 yr with family history or features suggestive of hereditary pheochromocytoma or for patients with sympathetic paragangliomas. For individuals who do not meet these criteria, genetic testing is optional.

Hereditary medullary thyroid carcinoma (MTC) is caused by autosomal dominant gain-of-function mutations in the RET proto-oncogene. Associations between specific RET mutations (genotype) and the aggressiveness of MTC and presence or absence of other endocrine neoplasms (phenotype) are well documented. Mutations in six exons (10, 11, 13, 14, 15, and 16) located in either cysteine-rich or tyrosine kinase domains cause one of three distinctive clinical subtypes: familial MTC, multiple endocrine neoplasia (MEN) type 2A (including variants with Hirschsprung's disease and cutaneous lichen amyloidosis), and MEN 2B. Hallmarks of MEN 2A include MTC, pheochromocytoma, and hyperparathyroidism. MEN 2B is associated with an earlier onset of MTC and pheochromocytoma, the absence of hyperparathyroidism, and the presence of striking physical stigmata (e.g., coarse facies, ganglioneuromatosis, and marfanoid habitus). Familial MTC is not associated with other endocrine neoplasms; however, the accurate distinction between familial MTC and MEN 2A may be difficult in kindreds with small size, incomplete histories, or a predominance of young individuals who may not have yet fully manifested the syndrome. Genetic testing detects greater than 95% of mutation carriers and is considered the standard of care for all first-degree relatives of patients with newly diagnosed MTC. Recommendations on the timing of prophylactic thyroidectomy and the extent of surgery are based upon a model that utilizes genotype- phenotype correlations to stratify mutations into three risk levels.

Prior experience in kindreds with a codon 891 RET protooncogene mutation indicates that carriers of this mutation develop only hereditary medullary thyroid carcinoma without evidence of other manifestations of multiple endocrine neoplasia type 2. In this paper, we report the first documented case in which medullary thyroid carcinoma and pheochromocytoma were clinically expressed in members of a family affected by the codon 891 RET mutation. Genetic analysis of the RET protooncogene in this family revealed an exon 15 missense mutation at codon 891 that resulted in a serine to alanine amino acid substitution. These findings indicate that patients with this mutation should be screened for pheochromocytoma.

Hereditary medullary thyroid carcinoma, a tumor that arises from the parafollicular cells of the thyroid gland, occurs in isolation (as in familial medullary thyroid carcinoma), in association with hyperparathyroidism and pheochromocytoma (as in multiple endocrine neoplasia type 2A), or in association with pheochromocytoma, marfanoid habitus, and mucosal neuromas (as in multiple endocrine neoplasia type 2B). These genetic syndromes are associated with germline-activating mutations of the RET protooncogene, a cell surface tyrosine kinase receptor, which is believed to modulate specific intracellular signaling pathways involved in the regulation of C cell proliferation and apoptosis. RET-activating mutations involve two important functional areas of the receptor: the cysteine-rich extracellular domain and the intracellular tyrosine kinase domain. Multiple endocrine neoplasia type 2A and familial medullary thyroid carcinoma are more commonly associated with mutations in the cysteine-rich extracellular domain, whereas multiple endocrine neoplasia type 2B is exclusively associated with mutations involving the second intracellular tyrosine kinase domain. Here, we describe a novel missense mutation of the RET protooncogene that substitutes arginine for proline at codon 912 of the intracellular tyrosine kinase domain in a family with medullary thyroid carcinoma.

Calcitonin (CT) is a known inhibitor of bone resorption. Calcitonin gene-related peptide-alpha (CGRPalpha), produced by alternative RNA processing of the CT/CGRP gene, has no clearly defined role in bone. To better understand the physiologic role of the CT/CGRP gene we created a mouse in which the coding sequences for both CT and CGRPalpha were deleted by homologous recombination. The CT/CGRP(-/-) knockout (KO) mice procreated normally, there were no identifiable developmental defects at birth, and they had normal baseline calcium-related chemistry values. However, KO animals were more responsive to exogenous human parathyroid hormone as evidenced by a greater increase of the serum calcium concentration and urine deoxypyridinoline crosslinks, an effect reversed by CT and mediated by a greater increase in bone resorption than in controls. Surprisingly, KO mice have significantly greater trabecular bone volume and a 1.5- to 2-fold increase in bone formation at 1 and 3 months of age. This effect appears to be mediated by increased bone formation. In addition, KO mice maintain bone mass following ovariectomy, whereas wild-type mice lose approximately one-third of their bone mass over 2 months. These findings argue for dual roles for CT/CGRP gene products: prevention of bone resorption in hypercalcemic states and a regulatory role in bone formation.

Peripheral inflammation induced with a knee joint injection of a mixture of kaolin/carrageenan (k/c) produces primary and secondary hyperalgesia. Inflammatory pain is thought to involve a variety of transmitters released from nerve terminals, including amino acids, substance P (SP) and calcitonin gene-related peptide (CGRP). In the present study, mice deficient in the calcitonin/αCGRP gene (CGRP(−/−)) displayed normal responses to noxious stimuli. However, the CGRP knockout mice failed to demonstrate development of secondary hyperalgesia after induction of knee joint inflammation in two tests that assess central sensitization, through testing at sites remote from the primary insult. Nociceptive behavioral responses were assessed using the hot-plate test and paw withdrawal latency (PWL) to radiant heat applied to the hindpaw. The CGRP(−/−) mice showed no signs of secondary hyperalgesia after development of knee joint inflammation, while the expected significant decrease in the PWL was observed in the CGRP(+/+) mice as control. The CGRP(−/−) mice also had a prolonged rather than a shortened response latency in the hot-plate test 4 h after knee joint injection of k/c. Immunohistological study showed that CGRP-like immunoreactivity (CGRP-LI) was absent in the spinal cord and dorsal root ganglia taken from the CGRP(−/−) mice. These results indicate that endogenous CGRP plays an important role in the plastic neurogenic changes occurring in response to peripheral inflammatory events including the development of nociceptive behaviors.

Publisher Summary Calcitonin is produced by the C (or calcitonin-producing) cells of the thyroid gland. The active principle was subsequently shown to be a 32 amino acid peptide, released in response to an increase in the plasma calcium concentration. The calcitonin gene family includes at least four separate genes. The first (CALC I or CGRP a) encodes calcitonin and a second peptide, calcitonin gene-related peptide (CGRP). Primary RNA transcript of second gene is processed to produce only CGRPb. The third is a pseudogene and is not expressed. The fourth encodes the peptide, amylin. Based on primary structure, the CTs can be divided into three groups: artiodactyl, including porcine, bovine, and ovine, which differ by four amino acids; primate/rodent, including human and rat calcitonins, which differ by two amino acids; and teleost/avian, including salmon, eel, goldfish, and chicken calcitonins, which differ by three amino acids. Calcitonin secretion is regulated by the extracellular calcium concentration. Increasing the extracellular calcium concentration results in increased secretion of CT. This effect is mediated by the interaction of calcium with the calcium-sensing receptor (CaR), a G-protein-coupled receptor expressed in high levels on the thyroidal C cell. In contrast to the parathyroid cell, where increasing calcium concentration activates the CaR and links to pathways that inhibit parathyroid hormone secretion, activation of this same receptor in the C cell causes increased secretion of CT. Calcitonin and its related peptides (CGRPa, CGRPb amylin, and adrenomedullin) are small peptides that exert their biological effect by binding to and activating a family of G-protein-coupled receptors. This receptor is expressed on osteoclasts, renal tubular cells, neurons in the central and peripheral nervous system, and other cell types. Several different isoforms of the CT receptor are produced by alternative RNA processing, which couple differently to downstream transduction pathways.

The differential diagnosis of hypoglycemia in the pediatric age group is broad (Table 55-1). In the neonatal period, transient hypoglycemia caused by hyperinsulinemia is common. It is, however, the rare newborn who presents with profound and prolonged hypoglycemia requiring substantial glucose replacement. When this is detected, it most commonly is caused by hyperinsulinism.

Analysis of peripheral blood or tumor DNA samples from 101 patients with apparent sporadic medullary thyroid carcinoma (MTC) was performed to assess the frequency of RET proto-oncogene mutations in this patient population. Peripheral blood and/or tumor DNA was amplified by polymerase chain reaction. DNA sequence or restriction enzyme analysis was performed to detect mutations of RET proto-oncogene codons 609, 611, 618, 620, 634, 768, and 918. Six of 101 patients with apparent sporadic MTC had peripheral blood DNA mutations more commonly associated with hereditary MTC. In 4 patients, these mutations led to the identification of previously unrecognized kindreds. The remaining 2 patients were examples of de novo mutations. A codon 918 mutation was found in 14 of 57 (approximately 25%) tumor DNA samples. Mutations were not identified in the remaining patients. In this large cancer center population, approximately 6% of patients with sporadic MTC carry peripheral blood DNA mutations, either inherited or de novo, more commonly associated with MEN 2A or familial MTC. Seven additional gene carriers were identified as a direct result of these studies, a 2-fold multiplying effect. We conclude routine application of RET proto-oncogene testing should be included in all cases of apparent sporadic MTC.

Context: Medullary thyroid carcinoma (MTC) is a cancer of the parafollicular C cells commonly caused by an inherited or acquired RET protooncogene mutation. Therapeutic resistance and recurrence of the disease imply the presence of cancer stem cells in MTC. Objective: In this study, we sought to identify and characterize cancer stem cell-like cells in MTC. Main Outcome Measures: The characterization of stem cell properties was performed using immunostaining, flow cytometry, sphere formation assay, rederivation assay, Western blotting, and quantitative RT-PCR of defined markers of neural stem and progenitor cells. The role of RET activation was assessed through RNA interference knockdown. Results: CD133 positivity was identified by immunostaining patient MTC. Flow cytometry confirmed a subpopulation of CD133 cells in two MTC cell lines. The CD133 cells could be expanded by sphere formation assay, passaged multiple times, and expressed neural progenitor markers -tubulin 3 and glial fibrillary acidic protein. The MZ-CRC-1 cell line, which harbors a M918T RET mutation, had greater CD133 cell numbers and sphere-forming ability than the TT cell line, which harbors the less active C634W mutation. Sphere formation was more dependent on RET activity than epidermal growth factor or fibroblast growth factor. Conclusion: Our data support the existence of cancer stem-like cells in MTC, which exhibit the features of self-renewal and of multiple lineage differentiation that is dependent on RET receptor activity. These findings may provide new insights to develop more promising therapy for MTC. Differential Ligand-Mediated Pituitary Somatostatin Receptor Subtype Signaling: Implications for Corticotroph Tumor Therapy Anat Ben-Shlomo, Herbert Schmid, Kolja Wawrowsky, Oxana Pichurin, Erika Hubina, Vera Chesnokova, Ning-Ai Liu, Michael Culler, and Shlomo Melmed (J Clin Endocrinol Metab, published October 9, 2009, 10.1210/jc.2009-1311) ABSTRACT Objective: Pituitary targeted pharmacotherapy for Cushing’s disease is challenging and ineffective. Unlike octreotide and lanreotide, the multisomatostatin receptor (SST) analog pasireotide that exhibits SST5 greater than SST2 binding affinity offers potential for treating Cushing’s disease. Because corticotroph cells express SST5 more abundantly than SST2, pasireotide likely exerts superior corticotroph action mainly through SST5. However, there is no direct evidence for this assumption, and moreover, the ligand effect on corticotroph SST2 is not known. Results: We used AtT20 mouse pituitary corticotroph tumor cells stably overexpressing SST2 or SST5 and TtT/GF mouse pituitary folliculostellate cells stably or transiently expressing SST receptors to examine ligand-receptor activation by SST2and SST5selective agonists. We show that pasireotide was more potent than either octreotide or somatostatin-14 in mouse corticotroph cells. Pasireotide potency is not affected by SST2 abundance, SST2 antagonist treatment, or octreotide cotreatment in SST2overexpressing cells. Pasireotide also does not induce SST2 internalization and attenuates octreotide or SRIF14-induced SST2 internalization only at superphysiological dose ranges. In contrast, octreotide attenuates pasireotide potency in SST5-overexpressing cells. Moreover, short exposure to pasireotide causes prolonged inhibition of forskolin or CRH-induced cAMP accumulation, in contrast to somatostatin-14and SST2-selective agonists that induced postwithdrawal cAMP rebound. Long-term pasireotide signaling effects are enhanced by SST5 overexpression. Conclusion: The results indicate that SST5 determines shortand long-term enhanced pasireotide action in corticotroph cells, whereas the ligand action on SST2 is negligible. Endocrine Reviews, December 2009, 30(7):926–934 edrv.endojournals.org 933Objective: Pituitary targeted pharmacotherapy for Cushing’s disease is challenging and ineffective. Unlike octreotide and lanreotide, the multisomatostatin receptor (SST) analog pasireotide that exhibits SST5 greater than SST2 binding affinity offers potential for treating Cushing’s disease. Because corticotroph cells express SST5 more abundantly than SST2, pasireotide likely exerts superior corticotroph action mainly through SST5. However, there is no direct evidence for this assumption, and moreover, the ligand effect on corticotroph SST2 is not known. Results: We used AtT20 mouse pituitary corticotroph tumor cells stably overexpressing SST2 or SST5 and TtT/GF mouse pituitary folliculostellate cells stably or transiently expressing SST receptors to examine ligand-receptor activation by SST2and SST5selective agonists. We show that pasireotide was more potent than either octreotide or somatostatin-14 in mouse corticotroph cells. Pasireotide potency is not affected by SST2 abundance, SST2 antagonist treatment, or octreotide cotreatment in SST2overexpressing cells. Pasireotide also does not induce SST2 internalization and attenuates octreotide or SRIF14-induced SST2 internalization only at superphysiological dose ranges. In contrast, octreotide attenuates pasireotide potency in SST5-overexpressing cells. Moreover, short exposure to pasireotide causes prolonged inhibition of forskolin or CRH-induced cAMP accumulation, in contrast to somatostatin-14and SST2-selective agonists that induced postwithdrawal cAMP rebound. Long-term pasireotide signaling effects are enhanced by SST5 overexpression. Conclusion: The results indicate that SST5 determines shortand long-term enhanced pasireotide action in corticotroph cells, whereas the ligand action on SST2 is negligible. Endocrine Reviews, December 2009, 30(7):926–934 edrv.endojournals.org 933

The TT cells are a continuous line of human C-cells derived from a medullary thyroid carcinoma. These cells produce large quantities of calcitonin and calcitonin gene-related peptide (CGRP) by the differential splicing of a single calcitonin gene transcript. We have used specific cDNA probes for calcitonin and CGRP to study the regulation of the calcitonin gene by dexamethasone, a synthetic glucocorticoid. Northern blot analysis of total cellular RNA isolated from the TT cells showed hybridization of the calcitonin probe to 3600- and 1000-base RNA species. The CGRP probe hybridized to 3600- and 1050-base RNA species. Dexamethasone treatment (10(-9) to 10(-5) M) of TT cells (for 6 days) caused a dosage-dependent increase in calcitonin mRNA levels and a decrease in CGRP mRNA levels. These findings were confirmed in time course studies where dexamethasone treatment (10(-6) M) caused a 2-13-fold increase in calcitonin mRNA and a 40-60% decrease in CGRP mRNA between 4 and 6 days of treatment; the effect was reversible after dexamethasone withdrawal. After excluding an effect of dexamethasone on calcitonin and CGRP mRNA stability, we have concluded that dexamethasone affects the splicing mechanism to favor production of calcitonin mRNA over CGRP mRNA.

We utilized the TT cell, a human C-cell line derived from a medullary thyroid carcinoma, to study the effects of 1,25-dihydroxyvitamin D3 on cell growth and expression of the calcitonin gene. The growth rate of cells treated for 8 days with 1,25-dihydroxyvitamin D3 did not differ significantly from control or 24,25-dihydroxyvitamin D3 treated cells. Total RNA was isolated, and calcitonin and calcitonin gene-related peptide (CGRP) levels were measured by hybridization. 1,25 D3 lowered calcitonin and CGRP mRNA levels in a time- and dose-dependent fashion; 24,25 D3 had no effect. Northern blots revealed a decrease in the mature mRNA as well as the common precursor forms, indicating a transcriptional effect of 1,25 D3.

There have been few studies of physiological importance on the regulation of somatostatin by hormones. We have studied the effect of the synthetic glucocorticoid dexamethasone on somatostatin production in the human medullary thyroid carcinoma TT cell line, a model for somatostatin production by the parafollicular cell. Dexamethasone inhibited somatostatin production in a dose-related manner with a maximal effect at a concentration of 10(-6) M. TT cells treated with dexamethasone (10(-6) M) showed an almost complete inhibition of somatostatin peptide production by 48 h of treatment. Molecular sizing chromatography demonstrated a decrease in both the probable somatostatin precursor (13,000 dalton) and the fully processed peptide. Analysis of mRNA content by hybridization revealed that dexamethasone also caused a decrease in detectable somatostatin mRNA. The hybridizable somatostatin mRNA decreased to approximately 50% of basal levels within 12 h of treatment. Northern blot hybridization showed a decrease in a single RNA species representing mature somatostatin mRNA. Dose-response experiments revealed inhibition of both peptide and mRNA at concentrations from 1 X 10(-8) to 1 X 10(-5) M dexamethasone. Four days after withdrawal from dexamethasone treatment, peptide and mRNA levels were higher than dexamethasone-treated controls. The sex steroid estradiol had no inhibitory effect on somatostatin production. These results suggest a potential regulator of somatostatin production and provide a system for the study of somatostatin gene regulation.

Treatment of the somatostatin (SRIF)-producing TT cell line with 1,25 dihydroxyvitamin D3 (1,25 D3) lowered intracellular SRIF mRNA and peptide concentration. In separate experiments, the cAMP analog 8-(4-chlorophenylthio)-cAMP stimulated a rapid increase in SRIF mRNA content of the TT cells and SRIF peptide secretion. To determine whether 1,25 D3 could inhibit either the transcriptional or secretory effects of cAMP, TT cells were pretreated with 1,25 D3 for 4 days followed by treatment with the cAMP analog. Pretreatment with 1,25 D3 inhibited the cAMP-mediated increase of SRIF mRNA, but had no effect on the secretory response. We conclude that the ability of 1,25 D3 to silence SRIF gene expression is more potent than cAMP enhancer activity but that 1,25 D3 has no effect on that portion of the cAMP-dependent pathway which regulates peptide secretion.