Hsa-miR-139-5p is a prognostic thyroid cancer marker involved in HNRNPF-mediated alternative splicing.

It is critical to identify biomarkers and functional networks associated with aggressive thyroid cancer to anticipate disease progression and facilitate personalized patient management. We performed miRNome sequencing of 46 thyroid tumors enriched with advanced disease patients with a median follow-up of 96 months. MiRNome profiles correlated with tumor-specific histopathological and molecular features, such as stromal cell infiltration and tumor driver mutation. Differential expression analysis revealed a consistent hsa-miR-139-5p downexpression in primary carcinomas from patients with recurrent/metastatic disease compared to disease-free patients, sustained in paired local metastases and validated in publicly available thyroid cancer series. Exogenous expression of hsa-miR-139-5p significantly reduced migration and proliferation of anaplastic thyroid cancer cells. Proteomic analysis indicated RICTOR, SMAD2/3 and HNRNPF as putative hsa-miR-139-5p targets in our cell system. Abundance of HNRNPF mRNA, encoding an alternative splicing factor involved in cryptic exon inclusion/exclusion, inversely correlated with hsa-miR-139-5p expression in human tumors. RNA sequencing analysis revealed 174 splicing events differentially regulated upon HNRNPF repression in our cell system, affecting genes involved in RTK/RAS/MAPK and PI3K/AKT/MTOR signaling cascades among others. These results point at the hsa-miR-139-5p/HNRNPF axis as a novel regulatory mechanism associated with the modulation of major thyroid cancer signaling pathways and tumor virulence.

Trabecular bone score in patients with liver transplants after 1 year of risedronate treatment.

The aim of this study was to analyse the effect of risedronate on Trabecular Bone Score in liver transplant patients with low bone mass, during 1-year follow-up. In this retrospective cohort study, trabecular bone score (TBS) was calculated from dual X-ray absorptiometry images of the lumbar spine (LS), collected from a prospective randomized open-label 1-year trial performed in liver recipient patients. A total of 89 patients with osteopenia or osteoporosis were randomized to receive RIS plus calcium and vitamin D3 or calcium and vitamin D3. TBS was low in both groups at baseline, 6 and 12 months. Baseline TBS at the LS showed degraded microarchitecture in 22.8% of patients, partially degraded in 40.3%, and normal values in 36.8% of the patients. After 1 year of treatment, no difference in TBS was observed between both groups. No correlations were found between bone mineral density (BMD) and TBS values at any follow-up time point. No relationship was found between BMD, TBS or immunosuppressive drugs with incidental fracture. No significant effect in TBS was observed in liver transplant patients treated with RIS or calcium and vitamin D3 after 1 year of follow-up. In these patients, the clinical usefulness of this new tool should be established.

Genomic and immune landscape Of metastatic pheochromocytoma and paraganglioma.

The mechanisms triggering metastasis in pheochromocytoma/paraganglioma are unknown, hindering therapeutic options for patients with metastatic tumors (mPPGL). Herein we show by genomic profiling of a large cohort of mPPGLs that high mutational load, microsatellite instability and somatic copy-number alteration burden are associated with ATRX/TERT alterations and are suitable prognostic markers. Transcriptomic analysis defines the signaling networks involved in the acquisition of metastatic competence and establishes a gene signature related to mPPGLs, highlighting CDK1 as an additional mPPGL marker. Immunogenomics accompanied by immunohistochemistry identifies a heterogeneous ecosystem at the tumor microenvironment level, linked to the genomic subtype and tumor behavior. Specifically, we define a general immunosuppressive microenvironment in mPPGLs, the exception being PD-L1 expressing MAML3-related tumors. Our study reveals canonical markers for risk of metastasis, and suggests the usefulness of including immune parameters in clinical management for PPGL prognostication and identification of patients who might benefit from immunotherapy.

Bone loss after heart transplant: effect of alendronate, etidronate, calcitonin, and calcium plus vitamin D3.

OBJECTIVE: To compare the effects of calcitonin, etidronate, and alendronate in preventing bone loss during the first 2 years after heart transplant. METHODS: A total of 222 heart transplant recipients (mean [SD] age, 52.4 [10] years, 85% male) were evaluated. Patients with normal bone mineral density (reference group, n = 102) received 1000 mg/d calcium plus 800 IU/d vitamin D3. The rest were assigned to 200 IU/d of calcitonin (n=42), 400 mg/d etidronate orally for 14 days quarterly (n = 33), or 10 mg/d alendronate (n = 45). All patients received calcium and vitamin D. Bone mineral density was assessed by dual-energy x-ray absorptiometry in the lumbar spine, the entire femur, and the femoral neck at baseline and 6, 12, and 24 months after transplant. RESULTS: At 2 years after transplant, bone mineral density in the lumbar spine had decreased in the reference group (-3.07%), calcitonin group (-0.93%), and etidronate group (-1.87%) but not in the alendronate group (+4.9%; P <.001). After 2 years, bone mineral density in the entire femur decreased in all groups (-3.2% in the reference group, -3.6% in the calcitonin group, -4.6% in the etidronate group, and -0.5% in the alendronate group) but bone loss was significantly lower in the alendronate group (P <.001). Bone mineral density in the femoral neck also decreased in all groups. The incidence of vertebral fractures did not differ among groups. Adverse events were similar between groups. CONCLUSIONS: Alendronate therapy in heart transplant recipients was associated with a significant increase in bone mineral density in the lumbar spine and less bone loss at the hip.

Comprehensive molecular analysis of immortalization hallmarks in thyroid cancer reveals new prognostic markers.

BACKGROUND: Comprehensive molecular studies on tumours are needed to delineate immortalization process steps and identify sensitive prognostic biomarkers in thyroid cancer. METHODS AND RESULTS: In this study, we extensively characterize telomere-related alterations in a series of 106 thyroid tumours with heterogeneous clinical outcomes. Using a custom-designed RNA-seq panel, we identified five telomerase holoenzyme-complex genes upregulated in clinically aggressive tumours compared to tumours from long-term disease-free patients, being TERT and TERC denoted as independent prognostic markers by multivariate regression model analysis. Characterization of alterations related to TERT re-expression revealed that promoter mutations, methylation and/or copy gains exclusively co-occurred in clinically aggressive tumours. Quantitative-FISH (fluorescence in situ hybridization) analysis of telomere lengths showed a significant shortening in these carcinomas, which matched with a high proliferative rate measured by Ki-67 immunohistochemistry. RNA-seq data analysis indicated that short-telomere tumours exhibit an increased transcriptional activity in the 5-Mb-subtelomeric regions, site of several telomerase-complex genes. Gene upregulation enrichment was significant for specific chromosome-ends such as the 5p, where TERT is located. Co-FISH analysis of 5p-end and TERT loci showed a more relaxed chromatin configuration in short telomere-length tumours compared to normal telomere-length tumours. CONCLUSIONS: Overall, our findings support that telomere shortening leads to a 5p subtelomeric region reorganization, facilitating the transcription and accumulation of alterations at TERT-locus.

Initial clinical and treatment patterns of advanced differentiated thyroid cancer: ERUDIT study.

Background: Up to 30% of differentiated thyroid cancer (DTC) will develop advanced-stage disease (aDTC) with reduced overall survival (OS). Objective: The aim of this study is to characterize initial diagnosis of aDTC, its therapeutic management, and prognosis in Spain and Portugal. Methods: A multicentre, longitudinal, retrospective study of adult patients diagnosed with aDTC in the Iberian Peninsula was conducted between January 2007 and December 2012. Analyses of baseline characteristics and results of initial treatments, relapse- or progression-free survival ((RP)FS) from first DTC diagnosis, OS, and prognostic factors impacting the evolution of advanced disease were evaluated. Results: Two hundred and thirteen patients (median age: 63 years; 57% female) were eligible from 23 hospitals. Advanced disease presented at first diagnosis (de novo aDTC) included 54% of patients, while 46% had relapsed from early disease (recurrent/progressive eDTC). At initial stage, most patients received surgery (98%) and/or radioiodine (RAI) (89%), with no differences seen between median OS (95% CI) (10.4 (7.3-15.3) years) and median disease-specific-survival (95% CI) (11.1 (8.7-16.2) years; log-rank test P = 0.4737). Age at diagnosis being <55 years was associated with a lower risk of death (Wald chi-square (Wc-s) P < 0.0001), while a poor response to RAI to a higher risk of death ((Wc-s) P < 0.05). In the eDTC cohort, median (RP)FS (95% CI) was of 1.7 (1.0-2.0) years after RAI, with R0/R1 surgeries being the only common significant favourable factor for longer (RP)FS and time to aDTC ((Wc-s) P < 0.05). Conclusion: Identification of early treatment-dependent prognostic factors for an unfavourable course of advanced disease is possible. An intensified therapeutic attitude may reverse this trend and should be considered in poor-performing patients. Prospective studies are required to confirm these findings.

Effect of early risedronate treatment on bone mineral density and bone turnover markers after liver transplantation: a prospective single-center study.

The aim of this study was to investigate the effect of risedronate (RIS) on bone loss and bone turnover markers after liver transplantation (LT). Patients with osteopenia or osteoporosis within the first month after LT were randomized to receive RIS 35 mg/week plus calcium 1000 mg/day and vitamin D(3) 800 IU/day (n = 45) or calcium and vitamin D(3) at same dosages (n = 44). Primary endpoint was change in bone mineral density (BMD) 6 and 12 months after LT. Secondary endpoints included changes in serum ß-CrossLaps (ß-CTX) and procollagen type 1 amino-terminal peptide (P1NP) and fracture rate. Spine X-rays were obtained at baseline and after 12 months. There was no significant difference in BMD changes between both treatment groups at any sites; either at 6 or 12 months. Spine BMD increased in both groups at 12 months vs. baseline (P = 0.001). RIS patients had a significant increase in intertrochanteric BMD at 12 months (P < 0.05 vs. baseline). Serum ß-CTX decreased in both groups (P < 0.01), with significant differences between groups at 3 months. No significant difference in vertebral fracture incidence was found. After 12 months, BMD improved at lumbar spine and did not change at hip in both groups. Significant differences between both groups were not found. Other factors (calcium and vitamin D replacement, early prednisone withdrawal) seem to have also positive effects in BMD.

Analysis of Telomere Maintenance Related Genes Reveals NOP10 as a New Metastatic-Risk Marker in Pheochromocytoma/Paraganglioma.

One of the main problems we face with PPGL is the lack of molecular markers capable of predicting the development of metastases in patients. Telomere-related genes, such as TERT and ATRX, have been recently described in PPGL, supporting the association between the activation of immortalization mechanisms and disease progression. However, the contribution of other genes involving telomere preservation machinery has not been previously investigated. In this work, we aimed to analyze the prognostic value of a comprehensive set of genes involved in telomere maintenance. For this study, we collected 165 PPGL samples (97 non-metastatic/63 metastatic), genetically characterized, in which the expression of 29 genes of interest was studied by NGS. Three of the 29 genes studied, TERT, ATRX and NOP10, showed differential expression between metastatic and non-metastatic cases, and alterations in these genes were associated with a shorter time to progression, independent of SDHB-status. We studied telomere length by Q-FISH in patient samples and in an in vitro model. NOP10 overexpressing tumors displayed an intermediate-length telomere phenotype without ALT, and in vitro results suggest that NOP10 has a role in telomerase-dependent telomere maintenance. We also propose the implementation of NOP10 IHC to better stratify PPGL patients.

CD133 Expression in Medullary Thyroid Cancer Cells Identifies Patients with Poor Prognosis.

CONTEXT: The identification of markers able to determine medullary thyroid cancer (MTC) patients at high-risk of disease progression is critical to improve their clinical management and outcome. Previous studies have suggested that expression of the stem cell marker CD133 is associated with MTC aggressiveness. OBJECTIVE: To evaluate CD133 impact on disease progression in MTC and explore the regulatory mechanisms leading to the upregulation of this protein in aggressive tumors. PATIENTS: We compiled a series of 74 MTCs with associated clinical data and characterized them for mutations in RET and RAS proto-oncogenes, presumed to be related with disease clinical behavior. RESULTS: We found that CD133 immunohistochemical expression was associated with adverse clinicopathological features and predicted a reduction in time to disease progression even when only RET-mutated cases were considered in the analysis (log-rank test P < 0.003). Univariate analysis for progression-free survival revealed CD133 expression and presence of tumor emboli in peritumoral blood vessels as the most significant prognostic covariates among others such as age, gender, and prognostic stage. Multivariate analysis identified both variables as independent factors of poor prognosis (hazard ratio = 16.6 and 2; P = 0.001 and 0.010, respectively). Finally, we defined hsa-miR-30a-5p, a miRNA downregulated in aggressive MTCs, as a CD133 expression regulator. Ectopic expression of hsa-miR-30a-5p in MZ-CRC-1 (RETM918T) cells significantly reduced CD133 mRNA expression. CONCLUSIONS: Our results suggest that CD133 expression may be a useful tool to identify MTC patients with poor prognosis, who may benefit from a more extensive primary surgical management and follow-up.

[Prevalence of deficient and insufficient vitamin D levels in a young healthy population]. / Prevalencia de concentraciones deficientes e insuficientes de vitamina D en una población joven y sana.

BACKGROUND AND OBJECTIVES: Recent studies have shown a high frequency of insufficient serum vitamin D levels in the general population, especially in the elderly and in individuals with osteoporosis. Data from the young adult population are scarce, but also reveal a high prevalence of vitamin D insufficiency and deficiency in this age group. The main reasons for this high prevalence seem to be poor dietary vitamin D intake and low sun exposure. The aim of the present study was to determine the prevalence of vitamin D insufficiency and deficiency in a young healthy population and its association with concentrations of calcium and parathyroid hormone and sun exposure. METHODS: We performed an observational, descriptive study in 116 subjects (38 men and 78 women aged 26.56 +/- 3.32 years), during the late spring and early summer of 2007. Fasting blood samples were obtained and levels of 25-hydroxivitamin D, intact parathyroid hormone, calcium, albumin and creatinine were measured. A questionnaire designed to assess sun exposure and sunshine protection during the previous 12 months was administered. RESULTS: The mean value of 25-hydroxivitamin D obtained was 24.58 +/- 6.98 ng/ml. The subjects were divided into three groups according to 25-hydroxivitamin D levels: deficient: < 20 ng/ml (27.58%); insufficient: 20-30 ng/ml (56.03%); and sufficient: > or = 30 ng/ml (16.37%). No statistically significant differences were found between the groups or the studied variables except for age in relation to vitamin D levels. CONCLUSIONS: Our study shows a high prevalence of vitamin D insufficiency in a young healthy population with no clear relationship with sun exposure or sunscreen protection. The low intake of food rich in vitamin D and the lack of food fortification combined with scarce effective sun exposure could account for the low serum levels of vitamin D in this population.

Does renal function improve after parathyroidectomy in primary hyperparathyroidism? / ¿Mejora la función renal tras la paratiroidectomía en el hiperparatirodismo primario?

INTRODUCTION: Primary hyperparathyroidism (PHPT) is a common endocrine disorder characterised by hypercalcaemia and parathormone increase. Decreased glomerular filtration rate (<60ml/min) continues to be a parathyroidectomy (PTX) criterion in asymptomatic PHPT. The influence of PTX on renal function evolution is the subject of debate. OBJECTIVE: To analyse the clinical, laboratory and histological characteristics of patients undergoing PHPT, as well as renal function evolution after PTX. MATERIAL AND METHODS: Retrospective study of 297 patients diagnosed with PHPT and referred to surgery in a single centre between 1998 and 2016. Laboratory parameters were determined at baseline, one week and one year after PTX. RESULTS: The Incidence of PTX was 38 cases/million/year. Mean age was 60±14 years and 80.5% of the patients were female. Approximately 65.3% were asymptomatic. Nephrolithiasis was the most common clinical finding (33%), followed by bone involvement (29.5%). PTX indications were: clinical symptoms (34.7%), hypercalcaemia>11.2mg/dl (27%), nephrolithiasis (13%), low bone mass (12%), age<50 years (11%) and decreased glomerular filtration rate<60ml/min (2.3%). For diagnostic localisation, spect-MIBI had a sensitivity of 92% and cervical ultrasound of 70%. A total of 94.3% of PHPT cases were due to a parathyroid adenoma. After PTX, normalisation of PHPT-related parameters was observed. We found a significant increase in serum creatinine levels (0.81 vs 0.85mg/dl, P<.001) from the first week post-PTX until the end of the first year. The renal function was only found to be significant in patients with glomerular filtration rate>60ml/min (baseline serum creatinine levels 0.77mg/dl vs serum creatinine levels after one year 0.81mg/dl, P<.001). CONCLUSIONS: PHPT was asymptomatic in most patients who underwent surgery. Hypercalcaemia and nephrolithiasis were the most common indications of parathyroidectomy in asymptomatic patients. MIBI scan was the most useful localisation method. Surgical treatment of PHPT is followed by renal function impairment, which persists after the first week post-PTX.

PheoSeq: A Targeted Next-Generation Sequencing Assay for Pheochromocytoma and Paraganglioma Diagnostics.

Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. As the list of related genes expands, genetic diagnosis becomes more time-consuming, and targeted next-generation sequencing (NGS) has emerged as a cost-effective tool. This study aimed to optimize targeted NGS in PPGL genetic diagnostics. A workflow based on two customized targeted NGS assays was validated to study the 18 main PPGL genes in germline and frozen tumor DNA, with one of them specifically directed toward formalin-fixed paraffin-embedded tissue. The series involved 453 unrelated PPGL patients, of whom 30 had known mutations and were used as controls. Partial screening using Sanger had been performed in 275 patients. NGS results were complemented with the study of gross deletions. NGS assay showed a sensitivity ≥99.4%, regardless of DNA source. We identified 45 variants of unknown significance and 89 pathogenic mutations, the latter being germline in 29 (7.2%) and somatic in 58 (31.7%) of the 183 tumors studied. In 37 patients previously studied by Sanger sequencing, the causal mutation could be identified. We demonstrated that both assays are an efficient and accurate alternative to conventional sequencing. Their application facilitates the study of minor PPGL genes, and enables genetic diagnoses in patients with incongruent or missing clinical data, who would otherwise be missed.

Pituitary stalk lesions: the Mayo Clinic experience.

CONTEXT: Pituitary stalk lesions have various etiologies, often not clinically apparent. Pathological samples from these lesions are rarely obtained, because of the critical location and function of the hypophyseal stalk. OBJECTIVES: The purpose of this study was to characterize the etiological spectrum of pituitary stalk lesions seen at Mayo Clinic Rochester over 20 years and to determine whether specific magnetic resonance imaging (MRI) characteristics could provide clinician guidance with regard to the etiology of infundibular lesions. DESIGN: A retrospective review of patients with pituitary stalk lesions seen at Mayo Clinic Rochester between 1987 and 2006 was conducted. Demographic, clinical presentation, imaging, laboratory, operative, and pathology data were reviewed and are reported using descriptive statistics. RESULTS: Of the 152 pituitary stalk lesions included, 49 (32%) were neoplastic, 30 (20%) were inflammatory, 13 (9%) were congenital anomalies, and 60 (39%) were of unclear etiology. Diabetes insipidus was diagnosed in 43 (28%) of the 152 patients, and 49 (32%) patients had at least one anterior pituitary hormone deficit. Secondary hypogonadism was the most common endocrine deficiency. Eleven of 13 congenital lesions were round in appearance and 5 of 7 patients with neurosarcoidosis confirmed by pathology had a uniformly thickened pituitary stalk on MRI. There were no statistically significant correlations between hypopituitarism and the pattern of enhancement or size of the lesion. CONCLUSIONS: Findings on MRI remain key in guiding the diagnosis of pituitary stalk lesions, particularly when used in conjunction with other clinical clues. There are no good imaging predictors for hypopituitarism, making clinical evaluation of all patients with pituitary stalk lesions crucial.