Cure and survival of sporadic medullary thyroid carcinoma following systematic preoperative calcitonin screening.

BACKGROUND: The improvement in outcome of sporadic medullary thyroid carcinoma (MTC) during the last decades remains controversial, even if a trend toward a better prognosis has been recently proposed. This study was aimed to determine the time trend cure and survival rates in sporadic MTC according to the use of systematic preoperative calcitonin screening. METHODS: Retrospective analysis of 178 sporadic MTC patients operated between 1980 and 2017 was performed. The impact of prognostic factors on cure and survival following the introduction of routine preoperative calcitonin screening in 2001 was evaluated according to the year of surgery. RESULTS: Since 2001, a significant decline of node-positive tumors (from 56.1 to 34.7%) and advanced stage at diagnosis (stage III/IV from 56.1 to 34.7%) occurred, with a concomitant significant increase in cure rate (64.5% vs 38.6%; p = 0.0012) and survival (p < 0.05). At univariate analysis, the cure was achieved more frequently in more recently operated patients (64.5% vs 38.6%; p = 0.0012), in disease staging I/II (86.5% vs 13.5%; p < 0.0001), in patients undergoing preoperative calcitonin screening (63.8% vs 23.5%; p < 0.0001) and in the absence of lymph node metastases (86.5% vs 13.5%; p < 0.0001). At multivariate analysis, only preoperative calcitonin screening and stage at diagnosis turned out to be significant independent prognostic factors for cure and survival. CONCLUSION: The outcome of sporadic MTC improved in the new millennium; diagnosis was achieved earlier, at a less advanced stage. Routine preoperative calcitonin screening may have contributed to improve cure and survival rates.

Long-Term Outcome After Surgery for Medullary Thyroid Carcinoma: A Single-Center Experience.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a rare C cells-derived tumor, with a hardly predictable long-term prognosis. This study was aimed to evaluate the predictive factors of cure and survival after surgery for MTC in a monocentric series. METHODS: A retrospective analysis of the long-term outcomes was assessed in 255 MTC patients operated between 1980 and 2015 at Padua University hospital. RESULTS: Sporadic MTC occurred in 65.1% and hereditary MTC in 34.9% of patients. At a median follow-up of 93 months (range 7-430), the cure rate was 56.8%. The overall 10-year survival was 84.4%, and MTC-related death rate was 15.3%. Patients who died because of MTC had a median age of 61 years (range 21-84) and were at stages III-IV in all cases; deaths occurred in 18% of sporadic MTC, 6% of MEN2a and 66.7% of MEN2b patients. None of the patients at stages I-II died because of the disease, but 17.7% had persistent/recurrent disease. Based on univariate analysis, age, gender, genetic variant, extent and year of surgery, tumor size, lymph-nodal metastases and tumor stage significantly affected cure and survival rates. At multivariate analysis, only patient- and tumor-related features (age, lymph-nodal status and stage) remained significant independent prognostic factors. CONCLUSIONS: Radical surgery is the only chance of definitive cure in MTC, but it is possible only at early stage; in advanced stages, even extensive surgery could not grant cure and prolonged survival. Stage, nodal metastases and age remain the main predictive factors for cure and survival.

Calcitonin measurement and immunoassay interference: a case report and literature review.

Calcitonin (CT) is currently the most sensitive serological marker of C-cell disease [medullary thyroid carcinoma (MTC) and C-cell hyperplasia]. Starting with a report on a case that occurred at our institution, this review focuses on trying to explain the reasons behind the poor specificity and sensitivity of the various CT immunoassays. A 15-year-old patient was referred to our institution in May 2014 for moderately elevated CT levels. Thyroid ultrasonography (US) documented a colloidal goiter. Secondary causes of the hypercalcitoninemia (hyperCT) were ruled out. The mismatch between the clinical picture and the laboratory results prompted us to search for other reasons for the patient's high CT levels, so we applied the heterophilic blocking tube (HBT) procedure to the patient's sera before the CT assay. Using this pretreatment step, his serum CT concentration dropped to <1 ng/L, as measured at the same laboratory. Measuring plasma CT has an important role in screening for C-cell disease, but moderately elevated serum CT levels need to be placed in their clinical context, bearing in mind all the secondary causes of C-cell hyperplasia and the possibility of laboratory interference, before exposing patients to the risks and costs of further tests.

Iodine status from childhood to adulthood in females living in North-East Italy: Iodine deficiency is still an issue.

PURPOSE: This survey aimed to assess iodine status in a female population at different ages, also investigating their eating habits. METHODS: We measured urinary iodine concentrations (UIC) in: 634 females at puberty and 361 fertile women in 246 of whom were considered also their children (134 daughters and 120 sons). All subjects completed a food frequency questionnaire. RESULTS: Median UIC decreased from childhood to adulthood (median UIC 107, 77 and 55 µg/l in the young girls, females at puberty and fertile women, respectively). Though using iodized salt improved iodine status in all groups, a significantly higher UIC was only noted in females at puberty. Milk consumption significantly increased UIC at all ages. In mother-child (both daughters and sons) pairs, the children's median UIC was nearly twice as high as their mothers' (UIC 115 vs. 57 µg/l). Milk consumption varied significantly: 56% of the mothers and 76% of their children drank milk regularly. The children (both daughters and sons) and mothers who drank milk had UIC ≥100 µg/l in 59 and 34% of cases, respectively, among the pairs who did not drink milk, 44% of the children and 19% of the mothers had UIC ≥100 µg/l. On statistical regression, 3.6% of the variability in the children's UIC depended on that of their mothers. CONCLUSIONS: Dietary iodine status declines from childhood to adulthood in females due to different eating habits. A mild iodine deficiency emerged in women of child-bearing age that could have consequences during pregnancy and lactation.

Synergistic antitumour activity of RAF265 and ZSTK474 on human TT medullary thyroid cancer cells.

Medullary thyroid cancer (MTC) is an aggressive malignancy responsible for up to 14% of all thyroid cancer-related deaths. It is characterized by point mutations in the rearranged during transfection (RET) proto-oncogene. The activated RET kinase is known to signal via extracellular signal regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K), leading to enhanced proliferation and resistance to apoptosis. In the present work, we have investigated the effect of two serine/threonine-protein kinase B-Raf (BRAF) inhibitors (RAF265 and SB590885), and a PI3K inhibitor (ZSTK474), on RET-mediated signalling and proliferation in a MTC cell line (TT cells) harbouring the RETC634W activating mutation. The effects of the inhibitors on VEGFR2, PI3K/Akt and mitogen-activated protein kinases signalling pathways, cell cycle, apoptosis and calcitonin production were also investigated. Only the RAF265+ ZSTK474 combination synergistically reduced the viability of treated cells. We observed a strong decrease in phosphorylated VEGFR2 for RAF265+ ZSTK474 and a signal reduction in activated Akt for ZSTK474. The activated ERK signal also decreased after RAF265 and RAF265+ ZSTK474 treatments. Alone and in combination with ZSTK474, RAF265 induced a sustained increase in necrosis. Only RAF265, alone and combined with ZSTK474, prompted a significant drop in calcitonin production. Combination therapy using RAF265 and ZSTK47 proved effective in MTC, demonstrating a cytotoxic effect. As the two inhibitors have been successfully tested individually in clinical trials on other human cancers, our preclinical data support the feasibility of their combined use in aggressive MTC.

The combination of RAF265, SB590885, ZSTK474 on thyroid cancer cell lines deeply impact on proliferation and MAPK and PI3K/Akt signaling pathways.

Papillary thyroid cancer (PTC) is the most frequent thyroid cancer entity, accounting for 88 % of cases. It may metastasize and loose iodine uptake capability, preventing any radioiodine or surgical treatment. The main gene altered in PTC is BRAF, which is found altered in over 50 % of cases. Moreover MAPK and PI3K/Akt pathways are greatly implicated in PTC development. Many target therapies for PTC are currently under investigation, unfortunately without the expected results. Aim of this study was to characterized the preclinical effectiveness of novel promising drugs, RAF265, SB590885 and ZSTK474 in 3 thyroid cancer cell lines (BCPAP, K1, 8505C). RAF265 and SB590885 target differentially BRAF, while ZSTK474 acts on PI3K. IC50 demonstrated high drug activities ranging from 0.1 to 6.2 µM, depending on drugs and cell type, while combination index revealed an interesting synergistic effect of combination regimen (RAF265 + ZSTK474 and SB590885 + ZSTK474) in almost all cell lines. Moreover this synergistic effect was particularly evident by Western blot, whereas dual MAPK and PI3K/Akt inhibition was detected. In addition, treating cells with SB590885 induced marked morphological changes, leading to massive vacuolization. This suggests an activation of apoptotic process, as underlined by Annexin V flow cytometry analysis. Also cell cycle was altered in treated cells, without evidence of a common pattern, but rather with a more specific effect relying on single drug or combination regimen used. Since beneficial effects of in vitro combination regimen (RAF265 + ZSTK474 and SB590885 + ZSTK474), it is recommended additional investigation. These data suggest the potential use of combination regimen in in vivo experiment or afterwards in human PTC.

Selenium Supplementation, Body Mass Composition, and Leptin Levels in Patients with Obesity on a Balanced Mildly Hypocaloric Diet: A Pilot Study.

BACKGROUND: Adequate levels of selenium (Se) have protective effects against several chronic diseases, such as obesity. The aim of this study was to assess the effect of Se supplementation in a selected group of patients with obesity. METHODS: This randomized prospective study included 37 overweight/obese individuals aged 18-65 years, who adopted a slightly hypocaloric diet for 3 months. An intervention group received 240 µg/day of L-selenomethionine for 3 months; a control group received a placebo. Clinical and biochemical parameters, body composition measurements, and the Psychological General Well-Being Index (PGWBI) questionnaire were tested at the beginning and end of the treatment. RESULTS: A comparison of the two groups showed a significant change in body composition, involving a decrease in body fat mass, between the baseline and the end of the follow-up, in the intervention group. Unlike the placebo group, the group given Se had a significant increase in lean body and muscle mass and a significant decrease in leptin levels after 3 months on diet. At the end of the follow-up, the group given Se scored higher on the PGWBI than those who did not. CONCLUSION: Se could reinforce the effects of diet for overweight and obesity. This work was registered in the ISRCTN registry with study ID ISRCTN6106073.

Epidemiology of Simultaneous Medullary and Papillary Thyroid Carcinomas (MTC/PTC): An Italian Multicenter Study.

Background: The concomitant presence of papillary thyroid cancer (PTC) and medullary TC (MTC) is rare. In this multicentric study, we documented the epidemiological characteristics, disease conditions and clinical outcome of patients with simultaneous MTC/PTC. Methods: We collected data of patients with concomitant MTC/PTC at 14 Italian referral centers. Results: In total, 183 patients were enrolled. Diagnosis was mostly based on cytological examination (n = 58, 32%). At diagnosis, in the majority of cases, both PTC (n = 142, 78%) and MTC (n = 100, 54%) were at stage I. However, more cases of stage II-IV were reported with MTC (stage IV: n = 27, 15%) compared with PTC (n = 9, 5%). Information on survival was available for 165 patients: 109 patients (66%) were disease-free for both PTC and MTC at the last follow-up. Six patients died from MTC. Median time to progression was 123 months (95% confidence interval (CI): 89.3-156.7 months). Overall, 45% of patients were disease-free after >10 years from diagnosis (125 months); this figure was 72.5% for PTC and 51.1% for MTC. Conclusions: When MTC and PTC are concurrent, the priority should be given to the management of MTC since this entity appears associated with the most severe impact on prognosis.

Novel Prognostic Factors Associated with Cell Cycle Control in Sporadic Medullary Thyroid Cancer Patients.

BACKGROUND: Medullary thyroid cancer (MTC) is a rare neuroendocrine-derived malignancy. It is represented by sporadic and familiar forms, and both can have RET oncogene mutations. Numerous markers can be used to define MTC; however, none is generally approved for predicting the outcome of sporadic MTC. AIM: The aim of this work was to analyze PTTG1/securin and Aurora kinase A expressions in MTC patients, both at the gene and protein levels, and to define their prognostic role in MTC assessing their association with lab and clinical parameters. PATIENTS AND METHODS: Seventy-one sporadic MTC human samples were analyzed for RET mutations and by qPCR for PTTG1 and AURKA (Aurora kinase A) expression. Ki-67 levels and western blot reactivity for PTTG1 and Aurora kinase A were also determined in a selected cohort of patients. RESULTS: RET somatic mutations were found in 48% of the patients (34/71). PTTG1 expression was statistically different among the groups with or without regional lymph node metastasis (p < 0.0001) and advanced stage disease (p < 0.01). PTTG1 and AURKA expressions were statistically higher than those of controls (p = 0.01 and p < 0.002, respectively). PTTG1 expression and Ki-67 levels were statistically different among the groups with remitted or persistent disease (p < 0.05 and p < 0.01, respectively). We found a significant correlation between the expressions of AURKA and PTTG1 (p < 0.0002, r = 0.5298) and between the expressions of PTTG1 and Ki-67 (p = 0.01). Ki-67 levels were statistically different among the groups with or without metastatic lymph nodes (p = 0.01) or distant metastases (p = 0.003). CONCLUSION: The presence of an altered expression of PTTG1 and AURKA is a negative prognostic factor associated with a more aggressive course of disease, such as an advanced stage or disease persistence. It emerges as a cell cycle process mediated by the 2 factors, in addition to the RET pathway, which can be altered in MTC patients.

Unique Case of a Large Indolent Medullary Thyroid Carcinoma: Time to Reconsider the Medullary Thyroid Adenoma Entity?

BACKGROUND: Medullary thyroid carcinoma (MTC) is a rare neuroendocrine cancer originating from parafollicular, calcitonin (Ctn)-producing C-cells. Prognosis correlates with primary tumor stage and Ctn levels. PATIENT: We describe a case of MTC involving a mass 7 cm in its largest dimension, associated with high Ctn concentrations (> 5,000 pg/mL), but normal carcinoembryonic antigen levels, and with no lymph nodes or distant metastases, in complete remission after thyroid surgery. The MTC had very peculiar histological features, with an expansive, noninfiltrating growth around the thyroid follicles, and no signs of invasion. These histopathological characteristics are reminiscent of the C-cell adenoma described in animals. The tumor also revealed an ossifying extracellular matrix unlike the classical amyloid. Despite the size of the tumor and the patient's high Ctn levels at diagnosis, the case described here reached complete remission after surgery. CONCLUSIONS: Further studies are needed to clarify the characteristics of MTC and better predict its behavior at diagnosis.

Efficacy of educational intervention to improve awareness of the importance of iodine, use of iodized salt, and dietary iodine intake in northeastern Italian schoolchildren.

OBJECTIVE: An educational program was conducted among school-aged children to improve their knowledge about iodine prophylaxis, their iodine status, and their dietary habits. METHODS: At the baseline (T0) and after 6 mo (T1), participants (970 at T0 and 949 at T1) answered questionnaires testing their knowledge about iodine prophylaxis and their eating habits. Urine samples were collected from a randomly selected subgroup of participants (313 at T0 and 312 at T1). RESULTS: From T0 to T1 there was a significant improvement in respondents' knowledge about iodine prophylaxis (from 44% to 70%), iodized salt consumption (from 78% to 84%), and median urine iodine concentrations (from 70 µg/L to 91 µg/L). Milk and iodized salt intakes were associated with a better iodine status per se, and more so when used simultaneously. Girls drank milk less often than boys did (daily in 52% and 59% of cases, respectively). Children of foreign origin ate sodium-rich food more often than Italians did. CONCLUSION: Educational intervention improved the children's knowledge about iodine prophylaxis and use of iodized salt. Consuming salt in addition to milk improves iodine status. Children of foreign origin have different eating habits.

Amiodarone-Induced Thyrotoxicosis: Differential Diagnosis Using 99mTc-SestaMIBI and Target-to-Background Ratio (TBR).

PURPOSE OF THE REPORT: Distinguishing between amiodarone-induced thyrotoxicosis (AIT) caused by excessive hormone synthesis (AIT-1) or by a destructive process (AIT-2) has important therapeutic implications, but is still difficult and debated. Tc-sestaMIBI thyroid scintigraphy (99m-STS) has been proposed as a tool for classifying the two forms. MATERIAL AND METHODS: 30 AIT patients (11 females and 19 males) who underwent 99m-STS were retrospectively assessed for the present study. For each patient, a target-to-background ratio (TBR) was obtained on planar images. The TBR was then correlated with the qualitative assessment of the scans and the final clinical diagnosis. RESULTS: Considering clinical response to treatment as the gold standard for differential diagnosis, 14 cases of AIT-1, 12 of AIT-2, and 4 mixed forms were identified. 99m-STS was able to qualitatively identify all the mixed forms, while 1/14 AIT-1 and 6/12 AIT-2 cases were misdiagnosed as mixed forms. When the quantitative index (the TBR) was compared with the final clinical diagnosis, ROC curve analysis enabled us to identify an IBR of 0.482 during 99m-STS as a cut-off capable of discriminating between AIT-1 and AIT-2, with 100% specificity and 91.7% sensitivity (P < 0.0001, area under the curve: 0.982). CONCLUSIONS: Taking the TBR into consideration, 99m-STS proved a very useful tool for distinguishing AIT-1 from AIT-2, and thus offering patients appropriate treatment as of their diagnosis. This approach can avoid pointless and potentially dangerous combined overtreatments, and may speed up the return to normal thyroid function, which is crucial in AIT patients suffering from heart disease.

The Hobnail Variant of Papillary Thyroid Carcinoma: Clinical/Molecular Characteristics of a Large Monocentric Series and Comparison with Conventional Histotypes.

BACKGROUND: The hobnail variant of papillary thyroid carcinoma (HPTC) has an aggressive behavior. The aims of this prospective study were to define the clinical/molecular characteristics of HPTC, and to compare them to those of conventional papillary thyroid carcinoma (PTC). METHODS: From 2010 to 2016, 25 cases of HPTC, characterized clinically and molecularly (BRAF, RAS, TERT promoter, and TP53 mutations), were compared to a series of 165 consecutive cases of PTC. All patients underwent total thyroidectomy and received radioactive iodine treatment. Follow-up was available for 19 HPTC patients. RESULTS: Among the HPTC patients, 64% had a hobnail component ≥30%, and 64% had multifocal disease. The mean tumor size was 30 mm; 96% of tumors were angio-invasive; 68% were N1, and 12% were M1; 58% harbored the BRAFV600E mutation, 12% had a mutation in the TERT promoter, 17% had a TP53 mutation, and not had a RAS mutation. At a mean follow-up of 39 months, 32% of patients had biochemical and/or structural disease. Tumor size was the only significant difference between patients with persistent disease and those with an excellent response (40 mm and 24 mm, respectively; p = 0.02). Compared to the PTC control group, the HPTC patients had larger tumors (30 mm vs. 16 mm; p < 0.001), more frequent lymph node involvement (68% vs. 38%; p = 0.01), and remote disease (16% vs. 3%; p < 0.0001), a similar prevalence of the BRAFV600E mutation (58% vs. 59%), a higher prevalence of TP53 mutations (17% vs. 1%; p < 0.05), and a worse outcome (structural/biochemical disease: 32% vs. 9%; p < 0.0001). CONCLUSIONS: HPTC is an aggressive variant, characterized by large tumor size, lymph node involvement, a tendency to metastasize, and a worse outcome.

EF24 (a Curcumin Analog) and ZSTK474 Emphasize the Effect of Cabozantinib in Medullary Thyroid Cancer.

XL184 is a small-molecule kinase inhibitor recently included in first-line systemic therapy for patients with advanced, progressive medullary thyroid cancer (MTC). EF24 is a curcumin analog with a high bioavailability, and ZSTK474 is an inhibitor of the phosphatidylinositol 3-kinase signaling pathway. We investigated the effect of these compounds, alone and in combination, in two rearranged during transfection (RET)-mutated TT and MZ-CRC-1 MTC cell lines and in six mostly RET wild-type human MTC primary cultures. Low IC50 values demonstrated the efficacy of the drugs, whereas the combination index revealed an important synergistic effect of combinations of XL184 + ZSTK474 and XL184 + EF24. Cell-cycle changes and the induction of apoptosis or necrosis were modulated by single compounds or combinations thereof. Both XL184 and EF24, alone or combined, were effective in reducing calcitonin secretion. Western blot and in-cell Western analysis showed that the compounds prompted a decrease in general reactivity to phosphorylated antibodies. Our data confirm XL184 alone as the reference drug for RET-mutated MTC, but we also demonstrated that EF24 alone is effective in inhibiting MTC cell viability. We tested the combinations XL184 + ZSTK474 and XL184 + EF24 too, finding that they act synergistically, irrespective of RET mutation status.

MiR-375 and YAP1 expression profiling in medullary thyroid carcinoma and their correlation with clinical-pathological features and outcome.

Medullary thyroid cancer (MTC) is a tumor marked by an indolent growth for which few prognostic factors and therapeutic strategies are actually available. Different studies have recently appraised well-differentiated thyroid cancers are characterized by a dysregulation in different microRNA sets; however, only few of them investigated the role of miRNA expression in MTCs. In this study, we have assessed the miR-375 expression in a series of 130 MTCs (104 are sporadic and 26 familial) with a median follow-up of 39 months (range 1-138) and then we have correlated our results with the clinical-pathological features and the patients' outcome.Moreover, we have appraised YAP1 (Yes-associated protein 1) immunohistochemical expression in the same MTC series and in 5 C-cells hyperplasia (CCH) samples as well. We observed a significant upregulation of miR-375 in all MTCs, when compared to the normal thyroid tissues. Besides, miR-375 expression was found to be closely linked to neoplastic size, a chance of thyroid capsule infiltration, the risk of lymph node metastasis, and the staging of the tumor. At the end of follow-up, only 10% (13/130) showed a tumor progression and a higher miR-375 expression was found to be closely linked to a worst patient' outcome. On the contrary, YAP1 immunohistochemical expression was sharply downregulated in tumors, whereas it was weakly expressed in CCHs. Our results suggest miR-375 plays a central role in MTC progression and, therefore, we seek following the idea that miR-375 pathway may be an effective target in novel MTC therapeutic strategies.

Prognostic Impact of miR-224 and RAS Mutations in Medullary Thyroid Carcinoma.

Little is known about the function of microRNA-224 (miR-224) in medullary thyroid cancer (MTC). This study investigated the role of miR-224 expression in MTC and correlated it with mutation status in sporadic MTCs. A consecutive series of 134 MTCs were considered. Patients had a sporadic form in 80% of cases (107/134). In this group, REarranged during transfection (RET) and rat sarcoma (RAS) mutation status were assessed by direct sequencing in the tumor tissues. Quantitative real-time polymerase chain reaction was used to quantify mature hsa-miR-224 in tumor tissue. RAS (10/107 cases, 9%) and RET (39/107 cases, 36%) mutations were mutually exclusive in sporadic cases. miR-224 expression was significantly downregulated in patients with the following: high calcitonin levels at diagnosis (p = 0.03, r = -0.3); advanced stage (p = 0.001); persistent disease (p = 0.001); progressive disease (p = 0.002); and disease-related death (p = 0.0001). We found a significant positive correlation between miR-224 expression and somatic RAS mutations (p = 0.007). Patients whose MTCs had a low miR-224 expression tended to have a shorter overall survival (log-rank test p = 0.005). On multivariate analysis, miR-224 represented an independent prognostic marker. Our data indicate that miR-224 is upregulated in RAS-mutated MTCs and in patients with a better prognosis and could represent an independent prognostic marker in MTC patients.

Frequency and Significance of Ras, Tert Promoter, and Braf Mutations in Cytologically Indeterminate Thyroid Nodules: A Monocentric Case Series at a Tertiary-Level Endocrinology Unit.

PURPOSE: The management of thyroid nodules of indeterminate cytology is controversial. Our study aimed to establish the frequency and significance of H-,K-,N-RAS, TERT promoter, and BRAF gene mutations in thyroid nodes of indeterminate cytology and to assess their potential usefulness in clinical practice. METHODS: H-,K-,N-RAS, TERT promoter and BRAF gene mutations were examined in a series of 199 consecutive nodes of indeterminate cytology referred for surgical excision. RESULTS: 69/199 (35%) were malignant on histopathological review. RAS mutations were detected in 36/199 (18%), and 19/36 cases (53%) were malignant on histological diagnosis. TERT promoter mutations were detected in 7/199 (4%) nodules, which were all malignant lesions. BRAF mutations were detected in 15/199 (8%), and a BRAF K601E mutation was identified in 2 follicular adenomas and 1 noninvasive follicular thyroid neoplasm with papillary-like nuclear features. Altogether, this panel was able to identify 48% of the malignant lesions, achieving a specificity, positive predictive value, and negative predictive value for malignancy of 85, 62, and 75%, respectively. CONCLUSION: The residual malignancy risk in mutation-negative nodes is 25%. These nodes still need to be resected, but mutation analysis could help to orient the appropriate surgical strategy.

Overexpression of L-Type Amino Acid Transporter 1 (LAT1) and 2 (LAT2): Novel Markers of Neuroendocrine Tumors.

BACKGROUND: 6-18F-fluoro-L-3,4-dihydroxyphenylalanine (18F-FDOPA) PET is a useful tool in the clinical management of pheochromocytoma (PHEO) and medullary thyroid carcinoma (MTC). 18F-FDOPA is a large neutral amino acid biochemically resembling endogenous L-DOPA and taken up by the L-type amino acid transporters (LAT1 and LAT2). This study was conducted to examine the expression of the LAT system in PHEO and MTC. METHODS: Real-time PCR and Western blot analyses were used to assess LAT1 and LAT2 gene and protein expression in 32 PHEO, 38 MTC, 16 normal adrenal medulla and 15 normal thyroid tissue samples. Immunohistochemistry method was applied to identify the proteins' subcellular localization. RESULTS: LAT1 and LAT2 were overexpressed in both PHEO and MTC by comparison with normal tissues. LAT1 presented a stronger induction than LAT2, and their greater expression was more evident in PHEO (15.1- and 4.1-fold increases, respectively) than in MTC (9.9- and 4.1-fold increases, respectively). Furthermore we found a good correlation between LAT1/2 and GLUT1 expression levels. A positive correlation was also found between urinary noradrenaline and adrenaline levels and LAT1 gene expression in PHEO. The increased expression of LAT1 is also confirmed at the protein level, in both PHEO and MTC, with a strong cytoplasmic localization. CONCLUSIONS: The present study is the first to provide experimental evidence of the overexpression in some NET cancers (such as PHEO or MTC) of L-type amino acid transporters, and the LAT1 isoform in particular, giving the molecular basis to explain the increase of the DOPA uptake seen in such tumor cells.

The PDCD4/miR-21 pathway in medullary thyroid carcinoma.

Programmed cell death 4 (PDCD4) is a tumor suppressor gene involved in tumorogenesis. MicroRNA-21 (miR-21) specifically targets PDCD4, and recent studies suggest that PDCD4 is also regulated by Akt (antiapoptotic regulator within phosphatidylinositol 3-kinase). Medullary thyroid carcinoma (MTC) is a rare neuroendocrine cancer, and disease stage at diagnosis represents the main prognostic indicator. A consecutive series of 64 MTCs was considered. REarranged during Transfection (RET) and rat sarcoma (RAS) mutation status was assessed by direct sequencing. Quantitative real-time polymerase chain reaction was used to quantify mature hsa-miR-21. PDCD4 and Ki-67 immunostaining was performed with an automated platform. Immunoblot analysis of PI3K/Akt pathway was done on thyroid tissues. MTCs were consistently associated with miR-21 up-regulation (P < .0016) and featured significant PDCD4 nuclear down-regulation. An inverse correlation emerged between miR-21 overexpression and PDCD4 down-regulation (P = .0013). At enrollment, high miR-21 levels were associated with high calcitonin levels (P = .0003), lymph node metastases (P = .001), and advanced stages (P = .0003). At the end of follow-up, high miR-21 levels were associated with biochemically persistent disease (P = .0076). At enrollment, instead, PDCD4 nuclear down-regulation was associated with high calcitonin levels (P = .04), more advanced stages of disease (P < .01), and persistent disease after the follow-up (P = .02). p-Akt was more expressed in RAS-mutated MTC than in nonmutated cancers and normal tissue. This study showed, in MTCs, that miR-21 regulates PDCD4 expression and also that the miR-21/PDCD4 pathway correlates with clinicopathological variables and prognosis. Further studies should investigate the role of miR-21 as a prognostic biomarker and the feasibility of using PDCD4-restoring strategies as a therapeutic approach to MTC.

High-throughput mutation profiling improves diagnostic stratification of sporadic medullary thyroid carcinomas.

Sporadic medullary thyroid carcinoma (MTC) harbors RET gene somatic mutations in up to 50 % of cases, and RAS family gene mutations occur in about 10 %. A timely and comprehensive characterization of molecular alterations is needed to improve MTC diagnostic stratification and design-tailored therapeutic approaches. Twenty surgically resected sporadic MTCs, previously analyzed for RET mutations by Sanger sequencing using DNA from formalin-fixed paraffin-embedded samples, were investigated for intragenic mutations in 50 cancer-associated genes applying a multigene Ion AmpliSeq next-generation sequencing (NGS) technology. Thirteen (65 %) MTCs harbored a RET mutation; 10 were detected at both Sanger and NGS sequencing, while 3 undetected by Sanger were revealed by NGS. One of the 13 RET-mutated cases also showed an F354L germline mutation in STK11. Of the seven RET wild-type MTCs, four cases (57.1 %) harbored a RAS mutation: three in HRAS (all Q61R) and one in KRAS (G12R). The three remaining MTCs (15 %) resulted as wild-type for all the 50 cancer-related genes. Follow-up was available in all but one RET-mutated case. At the end of follow-up, 7 of 12 (58 %) RET-mutated patients had relapsed, while the 4 RAS-mutated MTC patients were disease-free. Two of the three patients with MTC wild-type for all 50 genes relapsed during the follow-up period. Detection of mutations by NGS has the potential to improve the diagnostic stratification of sporadic MTC.