Copy number variations alter methylation and parallel IGF2 overexpression in adrenal tumors.

Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the IGF2/H19 locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying IGF2 overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conn's adenoma (CA, n=12), pheochromocytomas (PCC, n=10), adrenocortical benign tumors (ACBT, n=20), and adrenocortical carcinomas (ACC, n=20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the IGF2/H19 locus including the H19 imprinting control region were integratively analyzed. IGF2 overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the H19 ICR suggesting that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with IGF2 overexpression in adrenal tumors and that hypermethylation of the H19 ICR is a consequence thereof.

Contemporary review of large adrenal tumors in a tertiary referral center.

BACKGROUND: Large adrenal tumors (LATs, ≥6 cm) are uncommon and associated with malignancy in 25% of cases. Their surgical management remains debatable. The aim of the present report was to evaluate the current incidence, nature and management of LAT. PATIENTS AND METHODS: We carried out a retrospective review of LATs managed in a tertiary referral center (2002-2011). RESULTS: Eighty-one patients were included (out of a total of 750 with adrenal tumors, 11%). Nine patients had no surgical intervention (11%). Fifty-two LATs were malignant (64%): adrenocortical carcinoma (44%), metastasis (27%) and pheochromocytoma (21%). Patients with malignant tumors exhibited a poorer 5-year overall survival than those with benign tumors (53.4% versus 96.3%, p=0.001). Disease-related mortality was approximately 60%, 29% and 0% for those with metastasis, adrenal carcinoma and malignant pheochromocytoma, respectively. The recurrence rate was the same for the three malignant sub-groups (30%). CONCLUSION: LATs are rare and more frequently malignant than previously reported. Some are benign and do not require for surgical intervention. Surgical indication and approach should be tailored for each patient.

Preoperative 18F-FDG uptake is strongly correlated with malignancy, Weiss score, and molecular markers of aggressiveness in adrenal cortical tumors.

BACKGROUND: Adrenal incidentaloma are frequent in the general population. It can be difficult to diagnose adrenocortical carcinomas among them, even with the progress of imaging techniques. We studied the results of PET-FDG in the diagnosis of such tumours. METHODS: We studied patients referred to the Department of Endocrine Surgery at La Timone Hospital, Marseilles, France, between June 2006 and October 2010 for adrenal tumours. All patients underwent a complete work-up (biological tests and imagery), completed with PET-FDG. We compared the results of PET-FDG and molecular analysis with Weiss score and clinical follow-up. We calculated correlations with the Pearson test. RESULTS: A total of 51 patients were studied. We found that PET-FDG had a sensitivity of 95% and specificity of 97% for the diagnosis of adrenocortical carcinoma. The correlation between PET-FDG and Weiss score was 77% (P ≤ 0.0001). Molecular analyses were correlated as well with Weiss score and malignancy (P < 0.05). CONCLUSIONS: The nature of atypical adrenal masses can be difficult to define during preoperative investigations. For undetermined tumours smaller than 6 cm, characterization with PET-FDG can be one more diagnostic argument pointing to malignancy. It could potentially change the therapeutic strategy and surgical management. In our experience, molecular analyses are available after surgery and have less impact on the therapeutic strategy than PET-FDG. Preoperative PET-FDG can be an asset in the management of adrenal incidentaloma and adrenocortical carcinoma.

Radiation doses to cohabitants of patients undergoing radioiodine ablation for thyroid cancer: poor compliance with radiation protection guidelines but low radiation exposure.

BACKGROUND: The drive to reduce hospital stay after radioiodine remnant ablation in patients with thyroid cancer may increase the risk of radiation exposure to family members. The aim of this study was to evaluate the key determinants of dose exposure to familial members, with particular reference to the degree of adherence to current radiation safety guidelines. METHODS: All participants prospectively received our standard departmental oral and written safety instructions, with a mandatory 3-day restriction period. The postmicturition radiation levels of treated patients were measured (at 1-m distance) at the time of discharge using a portable radiometer. The radiation exposure of cohabitants was assessed with an optically stimulated luminescence-based personal dosimeter during the 3 days after hospital discharge. A questionnaire was used to assess the adherence of relatives/cohabitants to radiation safety guidelines. RESULTS: A total of 38 patients with thyroid cancer and 48 household members were included. At 48 h post therapy, the patient's median emission at 1-m distance was 13.4 µSv/h. The mean cumulative cohabitant exposure was 102 µSv (<50-1000). A positive correlation between cohabitant radiation exposure and the radiation level of the patient was observed (P=0.016). This correlation was absent when the recommended guidelines were followed (P=0.56). Only 17 household members (35.4%) strictly followed the recommended guidelines, but dose exposures exceeded 0.3 mSv in only four cases, in which a mean of between 5.8 and 9.5 h were spent in close proximity to the patient in the first 3 days, including sleeping with treated patients in half of the cases. CONCLUSION: Despite poor compliance with safety guidelines, a short-stay protocol respects current legislation, and is applicable to most patients treated with 3.7 GBq for radioiodine remnant ablation.

Heath-related quality of life in thyroid cancer patients following radioiodine ablation.

BACKGROUND: There is limited information about the medium to long-term health-related quality of life (QOL) in thyroid cancer patients after initial therapy and the existing studies suffer from limitations. The aim of the study was to assess the determinants of medium-term QOL after the initial therapy. METHODS: Following a total thyroidectomy, 88 thyroid cancer patients received either rhTSH or hypothyroid-assisted radioiodine ablation (RRA) using 3.7 GBq (100 mCi) of radioiodine. QOL evaluation of the patients using the validated Functional Assessment of Chronic Illness & Therapy (FACIT) was performed at the time of inclusion (t0) and later at the 9-month post-RRA (t1). RESULTS: 83 patients were eligible for the final evaluation. Medium-term FACIT scores were not statistically different between t0 and t1 patients. All but one domain of the QOL score was similar between t0 and t1. Using a multivariate analysis, only age and immediate postoperative QOL scores were found to be determinants of the overall medium term 9-month QOL scores. Analysis showed that 'high QOL levels' (baseline and 9-month) and 'no depression', 'low anxiety levels', were associated with '<45 yrs', 'men', 'partner', and 'rhTSH stimulation'. CONCLUSIONS: The use of radioiodine ablation does not seem to affect the medium term QOL scores of patients. Medium-term QOL is mainly determined by pre-ablation QOL. The assessment of baseline QOL might be interesting to evaluate in order to adapt the treatment protocols, the preventive strategies, and medical information to patients for potentially improving their outcomes.

Expression of somatostatin receptors, dopamine D2 receptors, noradrenaline transporters, and vesicular monoamine transporters in 52 pheochromocytomas and paragangliomas.

While somatostatin receptors (sst), through somatostatin-radiolabeled analogs, are used, mainly in second line, in the diagnosis and treatment of pheochromocytomas (PCC) and paragangliomas (PGL), the clinical significance of dopamine receptor subtype 2 (D2) in PCC/PGL is unknown. Indeed, radiolabeled dopamine (DA) analogs such as fluorine 18 ((18)F)-DA, used for positron emission tomography in PCC localization, are mainly correlated to the presence of noradrenaline transporter (NAT) and vesicular monoamine transporters (VMAT) but not to D2. The aim of this study was to quantitate D2 and sst expression in 52 PCC/PGL and to compare it with that of 35 gastroenteropancreatic neuroendocrine tumors (GEP-NETs). Quantitative RT-PCR of sst(1-3) and sst5, D2, NAT, VMAT1/2 was performed in all tumors, while immunohistochemistry analysis of sst2 and D2 was performed in seven tumors. D2 mRNA was expressed in all PCC/PGL. Mean expression was significantly higher in PCC/PGL than in GEP-NETs (4.8 vs 0.5 copy/copy ß-glucuronidase (Gus)). sst2 and sst(1) were expressed in most PCC/PGL, with sst(2)-dominant expression (mean mRNA: 1.6 vs 0.4 copy/copy ß-Gus). sst2 expression level was similar to that of GEP-NETs, whereas sst5 expression level was significantly lower (0.12 vs 0.78 copy/copy ß-Gus). Our study evidenced strong D2 mRNA expression in PCC and for the first time in PGL. PCC/PGL express sst2 mRNA at levels similar to those of GEP-NETs. New drugs can target ssts and D2 more efficiently than current somatostatin analogs. Moreover, transporters like NAT and VMAT1/2, could be co-targeted with sst, as a basis of new radionuclide compounds in the imaging and treatment of these tumors.

18F-FDG avidity of pheochromocytomas and paragangliomas: a new molecular imaging signature?

UNLABELLED: Our objective was to evaluate (18)F-FDG PET uptake in patients with nonmetastatic and metastatic chromaffin-derived tumors. METHODS: Twenty-eight consecutive unrelated patients with chromaffin tumors, including 9 patients with genetically determined disease, were studied. A combination of preoperative imaging work-up, surgical findings, and pathologic analyses was used to classify the patients into 2 groups: those with nonmetastatic disease (presumed benign, n = 18) and those with metastatic tumors (n = 10). (18)F-FDG PET was performed in all cases. Visual and quantitative analyses were individually graded for each tumor. Somatic mutations of the succinate dehydrogenase subunits B and D and Von-Hippel Lindau genes were also evaluated in 6 benign sporadic tumor samples. RESULTS: All but 2 patients showed significantly increased (18)F-FDG uptake on visual analysis. The maximum standardized uptake value (SUVmax) ranged from 1.9 to 42 (mean +/- SD, 8.2 +/- 9.7; median, 4.6) in nonmetastatic tumors and 2.3 to 29.3 (mean +/- SD, 9.7 +/- 8.4; median, 7.4) in metastatic tumors. No statistical difference was observed between the groups (P = 0.44), but succinate dehydrogenase-related tumors were notable in being the most (18)F-FDG-avid tumors (SUVmax, 42, 29.3, 21, 17, and 5.3). Succinate dehydrogenase and Von-Hippel Lindau-related tumors had a significantly higher SUVmax than did neurofibromatosis type 1 and multiple endocrine neoplasia type 2A syndrome-related tumors (P = 0.02). (18)F-FDG PET was superior to (131)I-metaiodobenzylguanidine in all metastatic patients but one. By contrast, (18)F-FDG PET underestimated the extent of the disease, compared with 6-(18)F-fluorodopa PET, in 5 patients with metastatic pheochromocytoma. However, succinate dehydrogenase mutations (germline and somatic) and functional dedifferentiation do not adequately explain (18)F-FDG uptake since most tumors were highly avid for (18)F-FDG. CONCLUSION: (18)F-FDG PET positivity is almost a constant feature of pheochromocytomas and paragangliomas. It may be considered a molecular signature of such tumors, although which aspect of the plethora of molecular changes associated with dedifferentiation, germline genetic defects, or the adaptive response to hypoxia is responsible for this characteristic requires further elucidation.

Molecular genetic study comparing follicular variant versus classic papillary thyroid carcinomas: association of N-ras mutation in codon 61 with follicular variant.

Although the follicular variant of papillary thyroid carcinoma (FVPTC) has been classified as a papillary cancer based on nuclear features, its follicular growth pattern and potential for hematogenous spread are more characteristic of follicular carcinoma. To gain insight into the biologic nature of FVPTC, we compared genetic alterations characteristic of papillary and follicular thyroid carcinomas in 24 FVPTCs and 26 classic PTC (CPTCs). In FVPTCs, we observed ras mutation in 6 of 24 cases (25%), BRAF mutation in 1 of 13 cases (7.6%), and ret rearrangement in 5 of 12 cases (41.7%). In CPTCs, we found ras mutation in no case, BRAF mutation in 3 of 10 cases (30%), and ret rearrangement in 5 of 11 cases (45%). One FVPTC exhibited simultaneous ras mutation and ret/PTC1 rearrangement, and one CPTC harbored simultaneous BRAF mutation and ret/PTC3 rearrangement. Based on these findings, we concluded that ras mutation correlates with follicular differentiation of thyroid tumors whereas ret activation is associated with papillary nuclei but not with papillary architecture. ret activation is not exclusive of ras or BRAF mutation, whereas ras and BRAF mutations are mutually exclusive. The implications of these results for follicular and papillary carcinogenesis are discussed.

A single-institution 25-year review of true parathyroid cysts.

BACKGROUND: Parathyroid cysts (PCs) are rare, and their origin is a subject of debate. They have been described as either functional, causing hyperparathyroidism, or non-functional in eucalcaemic patients. PATIENTS AND METHODS: We have performed a 25-year departmental review of PCs. Features studied included the clinical presentation and intra-operative findings, and a histological review was performed. Cases of cystic degeneration of parathyroid adenomas and pseudocystic change were excluded. RESULTS: Over 25 years, 22,009 thyroidectomies and 2,505 parathyroidectomies were performed in our department. Amongst these, 38 non-functional PCs were documented in 37 patients. The mode of presentation included incidental findings on routine chest x-ray, compressive symptoms or an asymptomatic palpable neck mass. Aspiration was the initial treatment in 14 patients and was curative in 10 of these. Four out of 14 patients underwent surgical procedures for recurrence of the cyst that occurred 6 to 48 months after aspiration. In 27 patients, surgery was performed and all identified PCs were localized in the inferior parathyroid glands. Histologically, the cyst wall consisted in associations of lymphoid, muscular, thymic, salivary, adipose and mesenchymal tissues. CONCLUSIONS: PCs are rare but should be included within the differential diagnosis of a neck lump. True PCs are non-functional. Pathological and immunohistochemical findings are suggestive of a branchial origin. Fine-needle aspiration may be curative and is diagnostic due to the characteristic appearance of the fluid and high PTH levels on assay.

A plasminogen-like protein, present in the apical extracellular environment of thyroid epithelial cells, degrades thyroglobulin in vitro.

The prothyroid hormone, thyroglobulin (Tg), is stored at high concentrations in the thyroid follicular lumen as a soluble 19S homo-dimer and as heavier soluble (27S and 37S) and insoluble (Tgm) forms. Follicular degradation of Tg may contribute to maintaining Tg concentrations compatible with follicle integrity. Here, we report on the presence of a plasminogen-like protein in the follicular lumen of normal human thyroids and its synthesis and apical secretion by cultured epithelial thyroid cells. Since all the main luminal forms of Tg are cleaved by this plasminogen-like protein, we suggest that it contributes to Tg degradation in the follicular lumen.

Thyroid follicular adenomas may display features of follicular carcinoma and follicular variant of papillary carcinoma.

Thyroid follicular adenomas (FA) are encapsulated tumors lacking vascular, capsular or lymphatic invasion and the typical nuclear features of papillary carcinoma (PC). However, some FA demonstrate nuclear atypia reminiscent of either follicular carcinomas (FC) or follicular variant of papillary carcinomas (FVPC), suggesting they may represent precursors of malignant transformation. We hypothesized that an objective evaluation of nuclear chromatin patterns could be used to define atypical follicular tumors (AFT) that are likely to be premalignant. To test this hypothesis, we used a computer-aided image analysis system to define the chromatin pattern of nuclei from thyroid tumors. To validate the system, we analyzed 3000 nuclei from 10 FA, 10 FC, and 10 FVPC samples and accurately distinguished between these classes of tumors. Then, we analyzed nine AFT and, in parallel, we analyzed the tumors for activating mutations of N2-RAS and over-expression of RET. The predominant chromatin pattern of AFT was of FA type in two cases, FC type in two cases, and PC type in three cases. One case contained similar numbers of FC and PC nuclei and one was comprised of a mixture of the three nuclear types. Neither RAS mutation nor RET overexpression were detected in FA. N2-RAS mutations were found in 33% of AFT, 20% of FC and 20% of FVPC without correlation with chromatin pattern. Over-expression of RET was detected in 45% of AFT, 20% of FC and 50% of FVPC and was correlated with PC nuclei. These results show that AFT are a heterogeneous group of tumors, containing genuine benign tumors and tumors that share morphological and molecular features with follicular and papillary carcinomas that might be precursors of both types of thyroid carcinomas.

Antigenicity and immunogenicity of the C-terminal peptide of human thyroglobulin.

Thyroglobulin (Tg) is cleaved into several peptides during thyroid hormone synthesis, an oxidative process. P40, an iodinated C-terminal peptide from human Tg, has a molecular weight of about 40 kDa and contains two hormonogenic sites. P40 is the smallest peptide that is still recognized by monoclonal antibodies from mice immunized with human Tg directed against its immunodominant region. Since P40 also contains several T-cell epitopes, it is a good candidate for studying the primary events involved in the process of hormone synthesis leading to thyroid autoimmunity. The present results show that P40 is recognized by Tg antibodies from patients with thyroid disorders and induces Tg antibodies in CBA mice. P40 may therefore be involved in the autoimmune process, thus providing a useful tool for diagnostic and therapeutic purposes.

Can sporadic medullary thyroid carcinoma be biochemically predicted? Prospective analysis of 66 operated patients with elevated serum calcitonin levels.

Measuring serum calcitonin (CT) in patients with thyroid diseases allows preoperative diagnoses of sporadic medullary thyroid carcinoma (MTC) and C-cell hyperplasia (CCH). The aim of this prospective study was to distinguish biochemically between CCH and MTC. Basal CT (bCT) was determined in 7276 consecutive patients referred for thyroid disease. Patients with recurrent, persistent, or familial MTC were excluded. When bCT was > 10 pg/ml a pentagastrin-stimulated CT (sCT) assay was performed. Patients were routinely operated on when bCT > 30 pg/ml or sCT > 100 pg/ml or when other indications for surgery were present. An extensive search for CCH or microscopic MTC was conducted by immunochemistry. Pathologic findings were correlated with the bCT and sCT values. In this study 66 patients were included. No morphologic alterations of C-cells were observed in 5 patients; 16 patients presented with CCH and 45 with MTC. Statistical analysis revealed a correlation of sCT and overall bCT with tumor size and staging (p <0.001). Considering cutoff values for bCT of < or = 30 pg/ml and for sCT of < or = 200 pg/ml, the positive predictive value of the test to detect MTC was 100% and the negative predictive value 63%. No patients with MTC at stage 2 to 4 had bCT <30 pg/ml or sCT <200 pg/ml. A bCT value of < or = 30 pg/ml or sCT < or = 200 pg/ml (or both) is highly predictive of MTC, requiring total thyroidectomy with lymph node dissection. Values of bCT <30 pg/ml and sCT <200 pg/ml do not distinguish between CCH and MTC at stage 1. In this case total thyroidectomy at least is recommended, and the role of nodal dissection might be discussed.