Clinical and transcriptomic characteristics of a novel SMARCD2 mutation that disrupts neutrophil maturation and function.

We report a novel case of SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) mutation successfully treated with hematopoietic stem cell transplantation. The female patient presented delayed cord separation, chronic diarrhea, skin abscesses, skeletal dysmorphisms, and neutropenia with specific granule deficiency. Analysis of the transcriptomic profile of peripheral blood sorted mature and immature SMARCD2 neutrophils showed defective maturation process that associated with altered expression of genes related to specific, azurophilic, and gelatinase granules, such as LTF, CRISP3, PTX3, and CHI3L1. These abnormalities account for the prevalence of immature neutrophils in the peripheral blood, impaired function, and deregulated inflammatory responses.

Oral Therapy for the Treatment of Transthyretin-Related Amyloid Cardiomyopathy.

The care of systemic amyloidosis has improved dramatically due to improved awareness, accurate diagnostic tools, the development of powerful prognostic and companion biomarkers, and a continuous flow of innovative drugs, which translated into the blooming of phase 2/3 interventional studies for light chain (AL) and transthyretin (ATTR) amyloidosis. The unprecedented availability of effective drugs ignited great interest across various medical specialties, particularly among cardiologists who are now recognizing cardiac amyloidosis at an extraordinary pace. In all amyloidosis referral centers, we are observing a substantial increase in the prevalence of wild-type transthyretin (ATTRwt) cardiomyopathy, which is now becoming the most common form of cardiac amyloidosis. This review focuses on the oral drugs that have been recently introduced for the treatment of ATTR cardiac amyloidosis, for their ease of use in the clinic. They include both old repurposed drugs or fit-for-purpose designed compounds which bind and stabilize the TTR tetramer, thus reducing the formation of new amyloid fibrils, such as tafamidis, diflunisal, and acoramidis, as well as fibril disruptors which have the potential to promote the clearance of amyloid deposits, such as doxycycline. The development of novel therapies is based on the advances in the understanding of the molecular events underlying amyloid cardiomyopathy.

Paediatric MAS/HLH caused by a novel monoallelic activating mutation in p110δ.

This study provides evidence for the first time for APDS-1 presenting as MAS/HLH, with evident clinical implications in patient's management and prognosis.

Intracisternal delivery of PEG-coated gold nanoparticles results in high brain penetrance and long-lasting stability.

BACKGROUND: The increasing use of gold nanoparticles (AuNPs) in the field of neuroscience instilled hope for their rapid translation to the clinical practice. AuNPs can be engineered to carry therapeutics or diagnostics in the diseased brain, possibly providing greater cell specificity and low toxicity. Although there is a general enthusiasm for these tools, we are in early stages of their development. Overall, their brain penetrance, stability and cell specificity are critical issues that must be addressed to drive AuNPs to the clinic. RESULTS: We studied the kinetic, distribution and stability of PEG-coated AuNPs in mice receiving a single injection into the cisterna magna of the 4th ventricle. AuNPs were conjugated with the fluorescent tag Cy5.5 (Cy5.5-AuNPs) to track their in vivo distribution. Fluorescence levels from such particles were detected in mice for weeks. In situ analysis of brains by immunofluorescence and electron microscopy revealed that Cy5.5-AuNPs penetrated the brain parenchyma, spreading in the CNS parenchyma beneath the 4th ventricle. Cy5.5-AuNPs were preferentially found in neurons, although a subset of resting microglia also entrapped these particles. CONCLUSIONS: Our results suggest that the ICM route for delivering gold particles allows the targeting of neurons. This approach might be pursued to carry therapeutics or diagnostics inside a diseased brain with a surgical procedure that is largely used in gene therapy approaches. Furthermore, this approach could be used for radiotherapy, enhancing the agent's efficacy to kill brain cancer cells.

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.

Longitudinal Characterization of Immune Response in a Cohort of Children Hospitalized with Multisystem Inflammatory Syndrome.

BACKGROUND: Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe complication of SARS-CoV-2 infection caused by hyperactivation of the immune system. METHODS: this is a retrospective analysis of clinical data, biochemical parameters, and immune cell subsets in 40 MIS-C patients from hospital admission to outpatient long-term follow-up. RESULTS: MIS-C patients had elevated inflammatory markers, associated with T- and NK-cell lymphopenia, a profound depletion of dendritic cells, and altered monocyte phenotype at disease onset, while the subacute phase of the disease was characterized by a significant increase in T- and B-cell counts and a rapid decline in activated T cells and terminally differentiated B cells. Most of the immunological parameters returned to values close to the normal range during the remission phase (20-60 days after hospital admission). Nevertheless, we observed a significantly reduced ratio between recently generated and more differentiated CD8+ T- and B-cell subsets, which partially settled at longer-term follow-up determinations. CONCLUSIONS: The characterization of lymphocyte distribution in different phases of MIS-C may help to understand the course of diseases that are associated with dysregulated immune responses and to calibrate prompt and targeted treatments.

Comparison of the diagnostic accuracy of combined elastosonography and BRAF analysis vs cytology and ultrasonography for thyroid nodule suspected of malignancy.

OBJECTIVE: Diagnosing thyroid nodules preoperatively using traditional diagnostic tools - ultrasonography (US) and cytology - still carries a considerable degree of uncertainty, and surgery is recommended for a far from negligible number of patients simply for diagnostic purposes. Thyroid elastosonography (USE) and BRAF analysis have recently proved useful in detecting thyroid malignancies. The aim of this study is to establish whether combining USE and BRAF testing ameliorates preoperative diagnosis of thyroid nodule candidates for intervention by conventional approaches, thereby avoiding the need for diagnostic surgical procedures. DESIGN AND PATIENTS: We retrospectively analysed the files of 155 consecutive patients with 164 nodules, all assessed by ultrasonography, cytology, USE and BRAF testing, who underwent thyroid surgery. RESULTS: Of the 164 nodules, 74 (45%) were benign and 90 (55%) were malignant at final histology. Combining ultrasonography and cytology identified 21 (13%) as benign, 93 (57%) as malignant or probably malignant and 50 (30%) as 'suspended' (when the combined test was not able to classify the node as benign or malignant) with a 99% sensitivity, 28% specificity, 63% PPV, 95% NPV and 67% accuracy. Combining USE and BRAF testing indicated that 59 (36%) were benign, 74 (45%) were malignant and 31 (19%) were in a 'suspended' category, with a 95% sensitivity, 74% specificity, 82% PPV, 93% NPV and 86% accuracy. CONCLUSIONS: In assessing thyroid nodules suspected of malignancy, the combined analysis of USE and BRAF is equally sensitive and more specific than conventional procedures, achieving more accurate preoperative diagnoses than US and cytology combined. USE and BRAF analysis for thyroid nodule evaluation might reduce the number of unnecessary surgical procedures.

An N-glycosylation hotspot in immunoglobulin κ light chains is associated with AL amyloidosis.

Immunoglobulin light chain (AL) amyloidosis is caused by a small, minimally proliferating B-cell/plasma-cell clone secreting a patient-unique, aggregation-prone, toxic light chain (LC). The pathogenicity of LCs is encrypted in their sequence, yet molecular determinants of amyloidogenesis are poorly understood. Higher rates of N-glycosylation among clonal κ LCs from patients with AL amyloidosis compared to other monoclonal gammopathies indicate that this post-translational modification is associated with a higher risk of developing AL amyloidosis. Here, we exploited LC sequence information from previously published amyloidogenic and control clonal LCs and from a series of 220 patients with AL amyloidosis or multiple myeloma followed at our Institutions to define sequence and spatial features of N-glycosylation, combining bioinformatics, biochemical, proteomics, structural and genetic analyses. We found peculiar sequence and spatial pattern of N-glycosylation in amyloidogenic κ LCs, with most of the N-glycosylation sites laying in the framework region 3, particularly within the E strand, and consisting mainly of the NFT sequon, setting them apart with respect to non-amyloidogenic clonal LCs. Our data further support a potential role of N-glycosylation in determining the pathogenic behavior of a subset of amyloidogenic LCs and may help refine current N-glycosylation-based prognostic assessments for patients with monoclonal gammopathies.

BRAF analysis by fine needle aspiration biopsy of thyroid nodules improves preoperative identification of papillary thyroid carcinoma and represents a prognostic factor. A mono-institutional experience.

BACKGROUND: The current preoperative diagnosis of a thyroid mass relies on microscopic evaluation of thyroid cells obtained by fine needle aspiration biopsy (FNAB). More recently, FNAB has been combined with molecular analysis to increase the accuracy of the cytological evaluation. In this mono-institutional prospective study, we evaluated whether the routine introduction of BRAF testing in thyroid FNAB could help ameliorate the preoperative recognition of papillary thyroid carcinoma (PTC) in "suspended" or malignant cytological categories. Moreover, we investigated the prognostic role of the BRAFV600E mutation in PTC. METHODS: BRAFV600E analysis was performed in thyroid FNAB from 270 patients classified into one of five cytological categories THY1, THY2, THY3, THY4, THY5. All subsequently underwent thyroidectomy±node dissection, from October 2008 to September 2009 in our Department. For each cytological category, we considered the definitive histological diagnosis of PTC and the presence of the BRAFV600E mutation. In 141 patients with a final tissue diagnosis of PTC, we correlated the presence of BRAFV600E with gender, age, histotype, TNM, size of the lesion, extracapsular extension, node metastases and multifocality. RESULTS: The prevalence of the BRAFV600E mutation, among PTCs at final tissue diagnosis, was 69%. It improved the FNAB diagnostic accuracy from 88% to 91%. The BRAFV600E mutation was correlated with older age, classical variant of PTC, advanced stages in patients > 45 years. CONCLUSIONS: BRAFV600E testing could play a role in improving the diagnostic accuracy of FNAB for PTC, representing a useful adjuvant tool in presurgical characterization of thyroid nodes in particular cases. There is an association between the BRAFV600E mutation and some clinico-pathological characteristics of PTC.

Bone Marrow Microenvironment in Light-Chain Amyloidosis: In Vitro Expansion and Characterization of Mesenchymal Stromal Cells.

Immunoglobulin light-chain amyloidosis (AL) is caused by misfolded light chains produced by a small B cell clone. Mesenchymal stromal cells (MSCs) have been reported to affect plasma cell behavior. We aimed to characterize bone marrow (BM)-MSCs from AL patients, considering functional aspects, such as proliferation, differentiation, and immunomodulatory capacities. MSCs were in vitro expanded from the BM of 57 AL patients and 14 healthy donors (HDs). MSC surface markers were analyzed by flow cytometry, osteogenic and adipogenic differentiation capacities were in vitro evaluated, and co-culture experiments were performed in order to investigate MSC immunomodulatory properties towards the ALMC-2 cell line and HD peripheral blood mononuclear cells (PBMCs). AL-MSCs were comparable to HD-MSCs for morphology, immune-phenotype, and differentiation capacities. AL-MSCs showed a reduced proliferation rate, entering senescence at earlier passages than HD-MSCs. The AL-MSC modulatory effect on the plasma-cell line or circulating plasma cells was comparable to that of HD-MSCs. To our knowledge, this is the first study providing a comprehensive characterization of AL-MSCs. It remains to be defined if the observed abnormalities are the consequence of or are involved in the disease pathogenesis. BM microenvironment components in AL may represent the targets for the prevention/treatment of the disease in personalized therapies.

Influence of physiological dietary selenium supplementation on the natural course of autoimmune thyroiditis.

OBJECTIVE: Our study aimed to investigate whether physiological doses of selenium (Se) influence the natural course of autoimmune thyroiditis (AIT). DESIGN AND PATIENTS: A total of 76 consecutive patients (65 F, 11 M, median 43, range 15-75 years) with AIT, normal or slightly elevated TSH and fT4 within the normal range were divided into two groups: Group 0 (30 cases) was given no treatment while Group 1 (46 cases) was treated with sodium selenite 80 µg/day as a single oral dose for 12 months. Thyroperoxidase and thyroglobulin autoantibodies (TPO-Ab; Tg-Ab), TSH, fT4 and urine iodine concentrations (UIC) were measured at baseline and after 6 and 12 months of follow-up. Thyroid ultrasonography (US) was performed at each follow-up point. Echogenicity was measured by histographic analysis of gray-scale pixels (gsp) ranging from 0 = black to 255 = white. RESULTS: Thyroid echogenicity decreased significantly in both groups after 6 months, but after 12 months, it had changed no more in Group 1, whereas it had dropped further in Group 0. No significant variation in TPO-Ab or Tg-Ab levels was observed between the two groups after 6 months, but both values decreased significantly after 12 months in Group 1, and five patients in this group became negative for TPO-Ab. TSH and FT4 showed no significant variations in either group. CONCLUSIONS: Dietary supplementation with physiological doses of Se seems to be effective in preventing a reduction in thyroid echogenicity after 6 months of treatment and in reducing TPO-Ab and Tg-Ab after 12 months, but does not modify TSH or FT4.

Activated Phosphoinositide 3-Kinase Delta Syndrome 1: Clinical and Immunological Data from an Italian Cohort of Patients.

Activated phosphoinositide 3-kinase delta syndrome 1 (APDS-1) is a recently described inborn error of immunity caused by monoallelic gain-of-function mutations in the PIK3CD gene. We reviewed for the first time medical records and laboratory data of eight Italian APDS-1 patients. Recurrent sinopulmonary infections were the most common clinical feature at onset of disease. Seven patients presented lymphoproliferative disease, at onset or during follow-up, one of which resembled hemophagocytic lymphohistiocytosis (HLH). Genetic analysis of the PIK3CD gene revealed three novel mutations: functional testing confirmed their activating nature. In the remaining patients, the previously reported variants p.E1021K (n = 4) and p.E525A (n = 1) were identified. Six patients were started on immunoglobulin replacement treatment (IgRT). One patient successfully underwent hematopoietic stem cell transplantation (HSCT), with good chimerism and no GVHD at 21 months post-HSCT. APDS-1 is a combined immune deficiency with a wide variety of clinical manifestations and a complex immunological presentation. Besides IgRT, specific therapies targeting the PI3Kδ pathway will most likely become a valid aid for the amelioration of patients' clinical management and their quality of life.

Iodine status in pregnancy: role of dietary habits and geographical origin.

OBJECTIVES: A study was conducted on iodine status during pregnancy and its dependence on dietary habits, racial and geographical origin, and time since arrival in Italy. DESIGN AND METHODS: We enrolled 322 consecutive pregnant women: 217 Italians, 62 Eastern Europeans and 43 from Northern and Central Africa. All women completed a food frequency questionnaire on their dietary habits. The urinary iodide concentration (UIC) was determined in spot morning urine samples. RESULTS: In the group as a whole, the median UIC was 83 microg/l; the UIC was < 50 in 33% and of 150 microg/l or more in 27%; it was significantly lower in Africans and Eastern Europeans than in Italians (medians 45 and 46 vs. 100 microg/l, respectively, P = 0.005). For the foreign women, there was a significant correlation between UIC and time since arrival in Italy (r = 0.22, P = 0.02). A significant link emerged between UIC and cow's milk intake (P = 0.0001). Iodine supplements were used by 40% of the women, and UIC were higher in those who did so than in those who did not (median 103 vs. 75 microg/l, P = 0.03), particularly if the latter did not drink milk (median 98 vs. 42 microg/l, P = 0.01). Multivariate analysis showed that milk was the only variable influencing UIC (OR 1.29, P = 0.0005). CONCLUSIONS: (i) Iodine levels are too low among pregnant women in our region, and particularly in foreign women. (ii) Cow's milk intake is their main source of iodine. (iii) Iodine supplementation is mandatory during pregnancy, particularly for women do not drink milk.

Galectin-3 cytotest in thyroid follicular neoplasia: a prospective, monoinstitutional study.

OBJECTIVE: To analyze the utility of galectin-3 (Gal3) immunohistochemistry (IHC) on preoperatively obtained fine needle aspiration biopsy (FNAB) in identifying the subgroup of follicular neoplasia (FN) patients who were candidates for thyroidectomy. STUDY DESIGN: This prospective, monoinstitutional study applied a standardized Gal3 immunostaining protocol (cell block specimens; Gal3 scores: G0 [no Gal3+ve IHC reaction], G1 [Gal3+ve thyrocytes < or = 10%], G2 [Gal3+ve thyrocytes > 10%) in 100 consecutive cytologically assessed FN. All patients underwent thyroidectomy, and the FNs were always histologically categorized (World Health Organization criteria). RESULTS: The overall malignancy rate was 15%. Gal3 expression in presurgical samples significantly correlated with the postoperative diagnosis (p < 0.0001). When all positive Gal3 cases were pooled together (G1+G2), the IHC test performed as follows: sensitivity = 80%; specificity = 86%; positive predictive value = 50%; negative predictive value = 96%. All the Gal3-G2 cases (presurgical cell block) showed postoperative evidence of malignancy. All 9 cases of papillary tumor expressed Gal3 in both cell block and postoperative histology. Among the 6 follicular cancers, the prevalence of Gal3 overexpression in the cell block was 50%. CONCLUSION: The cell block procedure applied to thyroid FNAB samples enables the Gal3 cytotest to be implemented usefully in the preoperative identification of those cases of FN postoperatively found to be malignant (also significantly reducing the number of inappropriate thyroidectomies). Strong Gal3 expression should prompt immediate surgical treatment.

Molecular characteristics in papillary thyroid cancers (PTCs) with no 131I uptake.

OBJECTIVE: Papillary thyroid cancers (PTCs) with no iodine uptake have an aggressive behaviour and a poor prognosis. The aim of our study was to characterize, at molecular level, a subset of PTC with no 131 iodine ((131)I) uptake. DESIGN AND METHODS: Forty-eight cancer tissues were divided into three groups: Group 1, 28 primary cancers; Group 2, 7 recurrences capable of trapping (131)I; and Group 3, 13 recurrences incapable of trapping (131)I. mRNA levels of thyroid genes (sodium/iodide symporter NIS, thyroglobulin, thyroperoxidase and pendrin) and glycolytic metabolism genes (GLUT-1, hexokinase I and II) and BRAF mutations were evaluated in the different groups. RESULTS: Cancers with no (131)I uptake had slightly reduced NIS, significantly reduced thyroglobulin (P < 0.01), thyroperoxidase (P = 0.01) and pendrin (P = 0.03) and significantly increased GLUT-1 (P = 0.01) gene expression levels; and a high frequency of BRAF mutations (77%). BRAF(V600E) mutation, in both primary and metastatic thyroid cancers, is associated with a marked drop in thyroperoxidase (29-fold) and pendrin (20-fold) expression and a considerable increase (five-fold) in GLUT-1 expression. CONCLUSIONS: (1) The loss of (131)I uptake in recurrences depends not only on a decrease in NIS gene, but possibly on a reduction in the molecules regulating its intracellular metabolism; (2) the high GLUT-1 gene expression supports the use of positron emission tomography with specific tracers in clinical management of such cancers; and (3) BRAF(V600E) point mutations may lead to less differentiated phenotypes, suggesting a worse prognosis.

RET genotypes in sporadic medullary thyroid cancer: studies in a large Italian series.

BACKGROUND: Highly discrepant data about the different distribution of RET germline single nucleotide polymorphisms (SNPs) among patients with sporadic medullary thyroid cancer (sMTC) and controls are available. DESIGN AND PATIENTS: In the present case-control study, a wide panel of seven RET SNPs has been tested in the largest sMTC series and in a matched control group. RESULTS: None of the investigated polymorphisms show a significantly different distribution in patients with sMTC when compared to controls. Twenty haplotypes and 57 genotypes were generated, and their association with the disease and with the clinical features were statistically evaluated. Interestingly, 14 genotypes were found to be unique to sMTC patients and 25 to controls. Two haplotypes and three genotypes, all including the intronic variants IVS1-126 and IVS14-24, were significantly associated with sMTC patients and with a higher tumour aggression. The functional activity of the only nonsynonymous RET variant (c.2071C > A, G691S) was tested for the first time. Interestingly, Western blot analyses showed that the fraction of Ret9-G691S protein located at the plasma membrane level was overrepresented when compared to Ret9-WT, suggesting facilitated targeting at the cell membrane for this variant. However, no transforming activity was shown in a focus formation assay on cells carrying the Ret9-G691S, against a possible oncogenic role of G691S variant. CONCLUSIONS: RET genotypes including two intronic RET variants were associated with the risk of developing sMTC and to more aggressive behaviour. Further studies are warranted to elucidate whether these RET genotypes are in linkage disequilibrium with another susceptibility gene or whether these variants could play a role in the genesis of sMTC per se.

MicroRNA profiles in familial and sporadic medullary thyroid carcinoma: preliminary relationships with RET status and outcome.

BACKGROUND: MicroRNAs (miRNAs) are involved in the pathogenesis of human cancers, including medullary thyroid carcinoma (MTC). The aim of this study was to test the hypothesis that different miRNA profiles are related to RET status and prognosis in patients with hereditary MTC (hMTC) and sporadic MTC (sMTC). METHODS: We analyzed the expression of nine miRNAs (miR-21, miR-127, miR-154, miR-224, miR-323, miR-370, miR-9*, miR-183, and miR-375) by quantitative real-time-polymerase chain reaction in 34 cases of sMTC, 6 cases of hMTC, and 2 cases of C-cell hyperplasia (CCH). We also analyzed the immunohistochemical expression of PDCD4, an miR-21 gene target. sMTC (n=34) was genotyped for somatic RET and RAS mutations. Disease status was defined on the basis of the concentration of serum calcitonin at the latest follow-up and other parameters as indicated in the results. RESULTS: MTC and CCH were both characterized by a significant overexpression of the whole set of miRNAs (the increase being 4.2-fold for miR-21, 6.7-fold for miR-127, 8.8-fold for miR-154, 6.6-fold for miR-224, 5.8-fold for miR-323, 6.1-fold for miR-370, 13-fold for miR-9*, 6.7-fold for miR-183, and 10.1 for miR-375, p<0.0001). PDCD4 expression was significantly downregulated in MTC samples, consistent with miR-21 upregulation. Significantly lower miR-127 levels were observed in sMTC carrying somatic RET mutations in comparison to sMTC carrying a wild-type RET. In sMTC and familial MTC, the miR-224 upregulation correlated with the absence of node metastases, lower stages at diagnosis, and with biochemical cure during follow-up. CONCLUSIONS: miRNAs are significantly dysregulated in MTC, and this dysregulation is probably an early event in C-cell carcinogenesis. miR-224 upregulation could represent a prognostic biomarker associated with a better outcome in MTC patients.

Combined RET and Ki-67 assessment in sporadic medullary thyroid carcinoma: a useful tool for patient risk stratification.

OBJECTIVE: Medullary thyroid carcinoma (MTC) derives from the parafollicular C cells, being sporadic in 75% of cases and familial in 25%, due to RET proto-oncogene germinal mutations. In sporadic forms, stage at diagnosis is the most important negative prognostic factor. The aim of this study was to evaluate the prognostic impact of molecular and immunohistochemical markers in sporadic MTC. DESIGN AND METHODS: We studied 60 patients with sporadic MTC. For each case, we sought RET somatic mutations in the primary cancer and in lymph node metastases. The primary cancer also underwent immunohistochemical examination for Ki-67. RESULTS: A somatic RET mutation was found in 38% of patients, being M918T in 52% of them. We observed a statistically significant association between RET mutations and male gender (P<0.01), tumor size (P<0.05), lymph nodes (P<0.05) and distant metastases (P<0.001), advanced stage (P<0.05), increased risk of persistent disease (P=0.01), and low overall survival (P<0.01). High Ki-67 levels were similarly associated with extra-thyroid spread (P<0.05), lymph nodes (P<0.05) and distant metastases (P<0.001), advanced stage (P=0.01), and low overall survival (P=0.01). Combining somatic RET analysis with Ki-67 assessment seems to be useful for increasing the specificity of Ki-67 assessment alone and identifying patients with a more aggressive cancer: in our series, only the patients who died during the follow-up had both a somatic RET mutation and a Ki-67 expression level >50 cells/mm(2). CONCLUSIONS: The combined evaluation of RET and Ki-67 could act as an adjuvant prognostic marker useful for ameliorating the initial risk stratification of patients with sporadic MTC.