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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

IMPORTANCE: Anaplastic thyroid carcinoma (ATC) historically has a 4-month median overall survival (OS) from time of diagnosis, with disease-specific mortality approaching 100%. The association between recent major advancements in treatment and OS has yet to be evaluated. OBJECTIVE: To evaluate rates of OS in patients with ATC over the last 2 decades. DESIGN, SETTING, AND PARTICIPANTS: Retrospective cohort study in a single tertiary care institution. Patients with histopathological confirmation of ATC from January 2000 to October 2019 were included and divided into 3 groups according to date of presentation: 2000-2013, 2014-2016, and 2017-2019. MAIN OUTCOMES AND MEASURES: Overall survival compared among different treatment eras and differing therapies, including targeted therapy, immunotherapy, and surgery. RESULTS: Of 479 patients (246 men [51%]; median age, 65.0 [range, 21.1-92.6] years) with ATC evaluated, 52 (11%) were stage IVA, 172 (36%) stage IVB, and 255 (53%) stage IVC at presentation. The median OS of the entire cohort was 0.79 years (9.5 months), ranging from 0.01 to 16.63. The OS at 1 and 2 years was 35% (95% CI, 29%-42%) and 18% (95% CI, 13%-23%) in the 2000-2013 group (n = 227), 47% (95% CI, 36%-56%) and 25% (95% CI, 17%-34%) in the 2014-2016 group (n = 100), and 59% (95% CI, 49%-67%) and 42% (95% CI, 30%-53%) in the 2017-2019 group (n = 152), respectively (P

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 (

Background The aim of this study was to investigate the genotype-phenotype relationship of pancreatic neuroendocrine tumors in patients with multiple endocrine neoplasia type 1 treated at our institution. Methods We conducted a retrospective chart review of all patients with multiple endocrine neoplasia type 1 treated at our center from January 1993 to December 2015. Presence of a pancreatic neuroendocrine tumor was determined based on imaging performed at any time from presentation to conclusion of follow-up. Results We reviewed 188 patients. The most common site of multiple endocrine neoplasia type 1 mutation was in exon 2 (34/188; 18%). Of 188 patients, 125 had a pancreatic neuroendocrine tumor (61%). Among all patients, 30 of 34 (88%) with an exon 2 mutation had a pancreatic neuroendocrine tumor compared with 95 of 154 (62%) with a mutation in exons 3–10 (P = .002). In the age group of 20 to 40 years, 8 of 9 patients with an exon 2 mutation had a pancreatic neuroendocrine tumor, compared with 24 of 52 patients (46%) with a mutation in exons 3–10 (P = .028). Patients with an exon 2 mutation had a greater frequency of pancreatic neuroendocrine tumor distant metastasis (53% vs 23%, P = .049). Conclusion Young patients with multiple endocrine neoplasia type 1 and an exon 2 mutation appear to have a 2-fold greater risk for developing a pancreatic neuroendocrine tumor, and patients with an exon 2 mutation may be at greater risk for developing distant metastasis. Consideration should be given to more intensive screening and more liberal application of primary operative intervention in this potentially high-risk group.

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.

Background: Historical data suggests differentiated thyroid cancer (DTC) patients (pts) with brain metastases (mets) have overall survival (OS) of approximately 1 year; other reports refer median OS of 33months. Little is known about the mutation profile these pts. The TCGA indicates a median of 1 mutation in pts with PTC, most of which were low risk cases. Methods: We retrospectively studied clinical characteristics including number of lesions, therapeutic modalities, OS and mutational profile of TC pts with brain mets at a single cancer center between 2013-2018. Cases were identified by ICD-9 & -10 codes for TC and secondary neoplasm of brain but excluded if a separate malignancy accounted for brain mets. Somatic mutations were tested by targeted next-generation sequencing in the majority of cases. Results: Of 105 cases reviewed, 52 met entry criteria. Median age at diagnosis of brain mets was 62.5 years (4-85years); 31/52 (59.6%) were males. DTC accounted for the majority of the pts, 37/52 (71%) [papillary thyroid cancer 55.8%(PTC; 29/52); poorly differentiated thyroid cancer (PDTC; 5/52) 9.6%; follicular thyroid cancer 5.8% (3/52); Hurthle cell 0/52]; anaplastic thyroid cancer (ATC) 21.1% (11/52) & medullary thyroid cancer 7.7% (4/52). Symptoms were present in 50%, predominantly weakness 9/26 (34.6%), headaches 6/26 (23.1%), altered mental status 5/26 (19.2%), visual changes 5/26 (19.2%) and seizures 4/26 (15.4%). 47 pts received systemic therapy as follows: antiangiogenic drug 26/47 (55%); BRAF inhibitor alone or in combination with MEK inhibitor 22/47 (46.8%) and checkpoint inhibitors 16/47 (34%). Molecular testing was available in 50 cases. Known oncogenic driver mutations were noted in 86%(43/50); BRAF V600E mutated in 25/50 (50%), RAS 14/50 (28%; NRAS n=11, HRAS n=2, KRAS n=1), RET mutated in 4 cases. Of 25 pts tested for TERT promoter mutation 8 (32%) were positive. Mean number +/- SD of somatic mutations was 2.14 +/- 1.15 in PTC; 2.6 +/-1.62 in PDTC; and 5.45 +/- 4.6 in ATC. In terms of number of brain lesions, 38.5% had ≥3 metastatic foci. Treatment consisted of radiation [stereotactic radiosurgery 22/50 (44%), whole brain radiation 10/50 (20%)] and 13/50 (26%) surgical intervention. Median OS was 29.7 months (95% CI 12.5-NR) after brain mets diagnosis. 1-year survival by number of brain metastases was 76% for single focus (n=17), 66% for 2 (n=15) and 50% (n=20) for ≥3 (p=0.034). One year OS for surgically treated vs radiation was not statistically different. 1-year OS for DTC was 65% (95% CI 51%, 82%) vs 53% in ATC (95% CI 30%, 94%) p-value=0.996. The OS in symptomatic vs silent brain mets was 19.9 months vs 29.6 months (p value= 0.45). Conclusions: Mutation burden appears to be higher in TC pts with brain mets compared to TCGA. In TC pts with brain mets, survival decreases as number of lesions increases. The OS was slightly shorter in pts with symptomatic brain mets but not statistically significant.

Background The cyclin-dependent-kinase inhibitor/retinoblastoma pathway has been implicated in sporadic medullary thyroid carcinoma tumorigenesis. Somatic CDKN2C loss has been associated with decreased overall survival in medullary thyroid carcinoma patients. We evaluated CDKN2C loss in a prospective clinical environment using a novel Clinical Laboratory Improvement Amendments–certified assay to confirm its association with aggressive disease and to interrogate response to targeted therapy. Methods Patients with advanced sporadic medullary thyroid carcinoma underwent tumor genotyping for the purpose of management of targeted therapy and prognostication. Results Of patients with informative CDKN2C assay results, 30 (51.8%) were haploinsufficient/1N and 28 (48.3%) were 2N. Forty patients (69.0%) had a somatic RET mutation, and 36.9% had alterations of both genes. Thirty patients (51.7%) were treated with systemic therapy. Presence of genetic alterations in CDKN2C or RET did not predict treatment response. Patients with 1N CDKN2C loss had significantly shorter time-to-distant-metastasis than patients with normal copy number (P = .03). Conclusion This is the first evaluation in the clinical setting of CDKN2C haploinsufficiency in sporadic medullary thyroid carcinoma. Although a larger cohort and longer follow-up will be required, loss seems to be associated with more aggressive disease and may indicate patients that might receive benefit from treatment with a CDK inhibitor.

Background Radiation-associated fusion oncogenes play a direct role in papillary thyroid cancer development and pathogenic fusions have recently been reported in medullary thyroid cancer. To date, no studies have evaluated oncogenic events in medullary thyroid cancer in a radiation-exposed population. Methods Somatic and germline alterations, including RET fusions, were evaluated in paired medullary thyroid cancer tumor and normal samples from the Chernobyl Tissue Bank, a heavily screened population affected by the Chernobyl disaster. Results Tissue was available for 49 individuals. The median age of diagnosis was 26 years (range 9 to 43 years); 16 were radiation-exposed at a median age of 6 (range 2 days to 17 years). A total of 21 patients harbored germline RET mutations (codons 634[13], 918[5], 790[1], 609[1], and 620[1]); 4 had family history. Sporadic medullary thyroid cancer was identified in 27 patients (RET[18], KRAS[1], RET+KRAS[1], TP53[1], wild type [6]), with 1 RET fusion (1/49;2%). The age at operation for patients with hereditary medullary thyroid cancer was not different than sporadic medullary thyroid cancer (23.5 vs 28 years, P = .063). In sporadic medullary thyroid cancer, radiation was not associated with a difference in age at operation, tumor size, or tumor stage (P > .05). Conclusion In a heavily screened cohort, genetic analysis revealed germline RET mutations in previously unrecognized probands and a remarkable number of sporadic medullary thyroid cancer cases with a young age at presentation.

Many advances have been made in the field of thyroid cancer for patients with locally advanced, unresectable and/or metastatic disease. There are now four anti-angiogenic multikinase inhibitors approved for medullary and differentiated thyroid cancer. However, as with other solid tumors, patients develop resistance to these targeted therapies or have contraindications to the approved therapies. Alternatives to the anti-angiogenic class of drugs are needed for these patients, thus, testing other drugs in thyroid cancer will be important in order to continue to advance the field. In May 2018, the FDA approved the combination of dabrafenib plus trametinib for BRAF V600E mutated anaplastic thyroid cancer, but eventually resistance ensues. Also, treatments for non-BRAF mutated patients are in great need. Gaining a better understanding of the mechanisms of resistance to targeted therapy will also be important in order to rationally modify treatment regimens. This chapter focuses on emerging treatments in clinical trials, translational studies that may one day lead to a better understanding of the mechanisms of resistance to targeted therapies and new strategies to identify patients suited for a particular genomic-driven therapy, as these will shape the future directions of thyroid cancer research.

Anaplastic thyroid cancer is a rare and aggressive thyroid cancer with an overall survival measured in months. Because of this poor prognosis and often advanced age at presentation, these patients have traditionally been treated palliatively and referred for hospice. However, recent progress using novel therapies has energized the field, and several promising clinical trials are now available for these patients. This review will highlight this progress and the potential treatments that could pave the way to improved outcomes and quality of life for patients with this disease.

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

Several guidelines for patients with multiple endocrine neoplasia 2A (MEN2A) take into account genotype and family history of medullary thyroid carcinoma (MTC) disease aggressiveness. We sought to determine if an association exists independent of genotype, which could provide important information for counseling MEN2A patients in management of their MTC. Pedigrees of patients with ≥5 family members with MEN2A were retrospectively reviewed. Analysis was performed among kindreds with the most frequently observed codon mutation (RET 634). Familial MTC disease aggressiveness was evaluated using: (1) mean age at diagnosis of MTC, (2) current mean age of carriers without MTC, (3) proportion of kindred with MTC with metastatic disease at diagnosis, (4) proportion of kindred with MTC with metastasis/death from MTC as worst outcome, and (5) proportion of kindred with disease progression. 170 affected patients from 12 different MEN2A kindreds met inclusion criteria. The number of affected family members available for study per kindred ranged from 8 to 43 individuals. A difference in mean age of MTC diagnosis was found in screened patients (p = 0.01); mean age of MTC-free patients did not differ (p = 0.93). No differences were noted among kindreds in disease stage at presentation, worst outcome, or progression; marked variation in these measures was noted within families. In conclusion, a difference in age of MTC diagnosis among different RET 634 kindreds was identified. In contrast, notable intra-familial variability in disease aggressiveness was observed. Based on these findings, we recommend counseling patients with codon 634 mutations that their MTC disease course cannot be predicted by that of their relatives.

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.

6023Background: Anaplastic thyroid cancer (ATC) is a rare malignancy characterized by rapid progression and median overall survival (OS) of less than 6 months. With the goal of improving ATC outcom...

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.

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor deriving from the thyroid parafollicular cell. Thyroidectomy continues to serve as the primary initial treatment for this cancer. Because standard cytotoxic chemotherapy has proven ineffective, reoperation and external beam radiation therapy had been the only tools to treat recurrences or distant disease. The discovery that aberrant activation of RET, a receptor tyrosine kinase, is a primary driver of MTC tumorigenesis led to clinical trials using RET-targeting tyrosine kinase inhibitors. The successes of those trials led to the approval of vandetanib and cabozantinib for treating patients with progressive or symptomatic MTC. The availability of these drugs, along with additional targeted therapies in development, requires a thoughtful reconsideration of the approach to treating patients with unresectable locally advanced and/or metastatic progressive MTC.

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.

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.

Objective. —Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant disorder. The 3 recognized subtypes include MEN 2A, characterized by medullary thyroid carcinoma (MTC), pheochromocytoma (pheo), and hyperparathyroidism (HPT); MEN 2B, by MTC, pheo, and characteristic stigmata; and familial MTC (FMTC), by the presence of MTC only. The purpose of this study was to establish the relationship between specific mutations and the presence of certain disease features in MEN 2 which could help in clinical decision making. Design. —Correlative survey study of 477 MEN 2 families. Setting. —Eighteen tertiary referral centers worldwide. Patients. —A total of 477 independent MEN 2 families. Main Outcome Measures. —Association between the position and type of germline mutation in the RET proto-oncogene and the presence or absence of MTC, pheo, HPT, and/or other features in a family. Results. —There is a statistically significant association between the presence of any mutation at a specific position (codon 634) and the presence of pheo and HPT. The presence of a specific mutation, CGC at codon 634, has yet to be associated with FMTC. Conversely, mutations at codons 768 and 804 are thus far seen only with FMTC, while codon 918 mutation is MEN 2B-specific. Rare families with both MEN 2 and Hirschsprung disease were found to have MEN 2-specific codon mutations. Patients with Hirschsprung disease presenting with such mutations should be monitored for the possible development of MEN 2 tumors. Conclusions. —This consortium analysis suggests that genotype-phenotype correlations do exist and, if made reliably absolute, could prove useful in the future in clinical management with respect to screening, surveillance, and prophylaxis, as well as provide insight into the genetic effects of particular mutations.

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.

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.

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.

In this issue of Surgery, Bugalho and colleagues from Portugal, present their experience with RET testing of patients with sporadic and familial medullary thyroid carcinoma (MTC). Although their targeted approach to RET testing for patients in whom the mutation status is unknown (presumed sporadic MTC) is not often practiced in this country (where sequencing of exons 10, 11, and 13 to 16 is commonly performed), their paper serves to emphasize the importance of ordering RET gene testing on all patients with MTC. At present, knowledge of the RET mutation status and disease extent is required to determine the correct operation for any patient with MTC. Therefore, all surgeons caring for patients with presumed sporadic or inherited MTC need to know how to obtain genetic testing of the RET proto-oncogene. Inherited MTC can occur as part of multiple endocrine neoplasia type 2 (MEN 2) syndrome. MEN 2 is an autosomal dominant inherited cancer syndrome with 3 main subtypes: MEN 2A, MEN 2B, and familial MTC (FMTC). These clinical subtypes differ from each other in the spectrum of endocrine involvement and the biologic behavior of MTC. MEN 2A is clinically defined by the presence of MTC and either pheochromocytoma or hyperparathyroidism (or both) in a single individual, or the presence of 2 or more tumor types in multiple relatives of a single family. MEN 2B is characterized by MTC and pheochromocytoma, without hyper

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.

Pancreatic endocrine tumors (PETs) occur in at least 50% of patients with multiple endocrine neoplasia type 1 (MEN1) and are the leading cause of disease-specific mortality. However, the timing and extent of surgery for MEN1-related PETs is controversial owing to the indolent tumor growth seen in most patients and the desire to avoid complications associated with insulin dependence. To help resolve this controversy, we retrospectively analyzed the clinical characteristics, surgical treatment, and clinical outcome of patients with MEN1-related PETs.All patients had histologic or radiographic confirmation of a PET in the setting of MEN1. Disease progression was defined radiographically as the development of new pancreatic tumors or distant metastases. Progression-free survival (PFS) and overall survival (OS) were used as the endpoints of this analysis.We identified 98 patients with MEN1, 55 (56%) of whom had PETs, including 27 women and 28 men with a median age of 37 years (range 8-69 years) at the time of diagnosis. Functioning PETs were present in 35 (64%) of 55 patients, and nonfunctioning tumors were present in 20 (36%). Pancreatic surgery was performed in 38 (69%) of the 55 patients; and the first operation included enucleation (n = 4), total pancreatectomy (n = 3), Whipple procedure (n = 4), and distal pancreatectomy (n = 27). The median size of the resected tumors was 2.8 cm (range 0.6-11.0 cm). Recurrent disease developed in the residual pancreas in 7 (20%) of 35 at-risk patients a median of 7.8 years after the first operation, and distant metastases occurred in 5 (14 %) of 36 surgically treated patients without distant metastasis (2 patients had distant metastases when surgery on the primary tumor was performed) at a median of 2.7 years following surgery. At last follow-up, 16 (29%) of 55 patients with PETs had died, 12 (22%) were alive with disease, 26 (47%) were alive without evidence of disease, and 1 (2%) was lost to follow-up. The median OS was 19.5 years (range 13-26 years) and was significantly longer for patients who had functioning PETs versus those with nonfunctioning tumors (P = 0.0007), for patients who underwent surgical resection of their PETs versus those who did not (P = 0.0043), and for patients with localized versus metastatic PETs at the time of diagnosis (P0.0001). Multivariate analysis revealed that younger age, hormonal function, and PET resection were independently associated with longer OS.Our data suggest that early diagnosis and surgical excision of MEN1-related PETs improves survival. However, translating these data into a surveillance strategy for the early detection of PETs is complex owing to the potential morbidity of pancreatic resection and the risk of long-term insulin dependence.

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.

Hypothesis: Multiple endocrine neoplasia type 1 (MEN 1) syndrome is an autosomal dominant disorder caused by germline mutations in the MEN1 gene and characterized by multiple endocrine tumors, most notably in the parathyroid glands, pituitary, and pancreas. The syndrome demonstrates variable expressivity and considerable genetic heterogeneity. Patient data were examined for possible associations between genotype and phenotype. Design: We reviewed recorded medical data from 1975 to 2001 on patients with MEN 1 and compared specific types and locations of MEN1 gene mutations with manifestations of the syndrome. Patients and Results: We identified 109 affected patients from 24 MEN 1 kindreds. The phenotypic expression of MEN 1 in affected individuals included hyperparathyroidism in 74%, pancreatic endocrine tumors in 51%, and pituitary tumors in 35%. Twelve of 14 insulinomas occurred in patients with pituitary tumors. Mutation analysis was completed in 14 of 24 kindreds (80 of the 109 patients). Mutations were most common in exons 2 (31%), 9 (15%), and 10 (23%). All 21 patients with frameshift mutations (and known pancreatic endocrine tumor status) had such tumors. Pituitary tumors were associated with frameshift mutations in exon 2. Conclusions: The type and location of MEN1 mutations may be associated with the phenotypic expression of specific tumors. Such information may assist in the genetic counseling and surveillance of at-risk patients. A specific genotype-phenotype correlation is unlikely because of the heterogeneity of the mutations in the MEN1 gene. Arch Surg. 2002;137:641-647

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.

Background: Most patients from typical multiple endocrine neoplasia type 1 (MEN 1) kindreds harbor mutations in the MEN-1 gene, MEN1 . We hypothesized that some patients with atypical endocrine neoplasia would also have mutations in MEN1 . Methods: DNA sequencing analysis of mutations in the coding region of MEN1 was performed with genomic DNA obtained from peripheral blood lymphocytes in a total of 21 patients who had: typical MEN 1 (n = 8), clinical features suggestive of MEN 1 but without a family history of endocrinopathy (n = 7), and atypical endocrine neoplasia and a family history of endocrinopathy suggestive of MEN 1 (n = 6). Results: All 8 patients with typical MEN 1 had mutations in MEN1 . None of the 7 patients with features of MEN 1 , but without a family history of endocrinopathy, had a MEN1 mutation. In contrast, 4 of 6 patients with atypical endocrine neoplasia that included components of MEN 1 and a family history of endocrinopathy had mutations in MEN1 , including 2 patients with pheochromocytoma. Conclusions: Genomic mutations in MEN1 may frequently be identified in patients with atypical endocrine neoplasia, especially in the setting of a family history of endocrinopathy. Atypical presentations of MEN 1 may include pheochromocytoma. (Surgery 1999; 126: 1097-104.)

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.

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.