Case Report - Multinodular goiter in a patient with Congenital Hypothyroidism and Bannayan-Riley-Ruvalcaba syndrome: the possible synergic role of TPO and PTEN mutation.

We report the case of a paediatric female patient affected by Bannayan-Riley-Ruvalcaba syndrome (BRRS) and congenital hypothyroidism (CH) with homozygous mutation of the TPO gene. She underwent total thyroidectomy at the age of seven years because of the development of a multinodular goiter. BRRS patients present an increased risk of benign and malignant thyroid disease since childhood because of inactivating mutation of PTEN, an onco-suppressor gene. Instead, homozygous mutations in the TPO gene can be associated with severe forms of hypothyroidism with goiter; previous studies have described cases of follicular and papillary thyroid cancer in CH patients with TPO mutation despite a perfectly controlled thyroid function with Levothyroxine therapy. To our knowledge, this is the first case that describes the possible synergic role of coexisting mutation of both TPO and PTEN in the development of multinodular goiter underlining the importance of a tailored surveillance program in these patients, especially during childhood.

Osteocalcin modulates parathyroid cell function in human parathyroid tumors.

Introduction: The bone matrix protein osteocalcin (OC), secreted by osteoblasts, displays endocrine effects. We tested the hypothesis that OC modulates parathyroid tumor cell function. Methods: Primary cell cultures derived from parathyroid adenomas (PAds) and HEK293 cells transiently transfected with the putative OC receptor GPRC6A or the calcium sensing receptor (CASR) were used as experimental models to investigate γ-carboxylated OC (GlaOC) or uncarboxylated OC (GluOC) modulation of intracellular signaling. Results: In primary cell cultures derived from PAds, incubation with GlaOC or GluOC modulated intracellular signaling, inhibiting pERK/ERK and increasing active ß-catenin levels. GlaOC increased the expression of PTH, CCND1 and CASR, and reduced CDKN1B/p27 and TP73. GluOC stimulated transcription of PTH, and inhibited MEN1 expression. Moreover, GlaOC and GluOC reduced staurosporin-induced caspase 3/7 activity. The putative OC receptor GPRC6A was detected in normal and tumor parathyroids at membrane or cytoplasmic level in cells scattered throughout the parenchyma. In PAds, the membrane expression levels of GPRC6A and its closest homolog CASR positively correlated; GPRC6A protein levels positively correlated with circulating ionized and total calcium, and PTH levels of the patients harboring the analyzed PAds. Using HEK293A transiently transfected with either GPRC6A or CASR, and PAds-derived cells silenced for CASR, we showed that GlaOC and GluOC modulated pERK/ERK and active ß-catenin mainly through CASR activation. Conclusion: Parathyroid gland emerges as a novel target of the bone secreted hormone osteocalcin, which may modulate tumor parathyroid CASR sensitivity and parathyroid cell apoptosis.

Malignancy risk in indeterminate thyroid nodules with Hürthle cells: role of autoimmune thyroiditis.

INTRODUCTION: Hürthle cells are modified follicular thyroid cells, whose development and proliferation have been related to different stimuli inducing cellular stress. Most thyroid aspirates containing Hürthle cells are classified as indeterminate, although the specific risk of malignancy for this subtype of atypia remains unclear. The aim of our study was to assess if the presence of Hürthle cells in indeterminate thyroid nodules correlates with the risk of malignancy. We further evaluated if this risk can be modified by the presence of an underlying Hashimoto's thyroiditis. MATERIALS AND METHODS: We retrospectively analyzed all indeterminate thyroid nodules that were surgically treated at our institution between January 2010 and March 2019. For each nodule, we inferred the presence of Hürthle cells in the cytological report. Cytological findings were then correlated with histological reports. RESULTS: 354 indeterminate thyroid nodules were included in the study. The rate of malignancy resulted significantly lower in nodules exhibiting Hürthle cells compared to those negative for this cellular pattern (11.4% vs 22.5%, p = 0.01). Although there was no difference in the rate of malignancy in the whole population according to the presence or absence of Hashimoto's thyroiditis (21.5 vs 18.5%, p = 0.63), the significantly lower prevalence of malignant lesions in nodules with Hürthle cells was confirmed only in the presence of a histologically documented Hashimoto's thyroiditis (6.2% vs 32%, p = 0.005). CONCLUSIONS: The finding of Hürthle cells in indeterminate thyroid nodules is associated with a low risk of malignancy in patients with an underlying Hashimoto's thyroiditis. The clinical management of these lesions may therefore be more conservative.

Impact of the 8th Edition of the AJCC-TNM Staging System on Estimated Cancer-Specific Survival in Patients Aged 45-54 Years at Diagnosis with Differentiated Thyroid Carcinoma: A Single Center Report.

BACKGROUND: The 8th edition of the American Joint Committee on Cancer (AJCC) staging system changed the age cutoff for risk stratification of differentiated thyroid carcinoma (DTC), downgrading patients between 45 and 54 years to stage I or II. The aim of our study was to assess cancer-specific survival (CSS) in patients aged 45-54 years, in order to document the prognostic capability of the last edition of the staging system. METHODS: We retrospectively reviewed the medical records of 172 patients that from January 1st, 2005, to May 31st, 2017, were diagnosed at our institution with DTC when aged 45-54 years. We restaged patients according to the 8th edition of the staging system and estimated CSS. RESULTS: 101 out of 172 patients (58.7%) were reallocated to a lower stage. Of the 101 downstaged patients, 88 (88.9%) showed a high or intermediate American Thyroid Association (ATA) risk of recurrence. We recorded no cancer-specific deaths. CONCLUSIONS: Risk of cancer-specific mortality in patients aged 45-54 years with DTC is low, supporting the prognostic capability of the 8th edition of the staging system. However, we recommend to consider carefully the significant proportion of patients at intermediate or high risk of recurrence in this group of patients.

Yes-Associated Protein 1 Is a Novel Calcium Sensing Receptor Target in Human Parathyroid Tumors.

The Hippo pathway is involved in human tumorigenesis and tissue repair. Here, we investigated the Hippo coactivator Yes-associated protein 1 (YAP1) and the kinase large tumor suppressor 1/2 (LATS1/2) in tumors of the parathyroid glands, which are almost invariably associated with primary hyperparathyroidism. Compared with normal parathyroid glands, parathyroid adenomas (PAds) and carcinomas show variably but reduced nuclear YAP1 expression. The kinase LATS1/2, which phosphorylates YAP1 thus promoting its degradation, was also variably reduced in PAds. Further, YAP1 silencing reduces the expression of the key parathyroid oncosuppressor multiple endocrine neoplasia type 1(MEN1), while MEN1 silencing increases YAP1 expression. Treatment of patient-derived PAds-primary cell cultures and Human embryonic kidney 293A (HEK293A) cells expressing the calcium-sensing receptor (CASR) with the CASR agonist R568 induces YAP1 nuclear accumulation. This effect was prevented by the incubation of the cells with RhoA/Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitors Y27632 and H1152. Lastly, CASR activation increased the expression of the YAP1 gene targets CYR61, CTGF, and WNT5A, and this effect was blunted by YAP1 silencing. Concluding, here we provide preliminary evidence of the involvement of the Hippo pathway in human tumor parathyroid cells and of the existence of a CASR-ROCK-YAP1 axis. We propose a tumor suppressor role for YAP1 and LATS1/2 in parathyroid tumors.

miR-126-3p contributes to parathyroid tumor angiogenesis.

Tumors of the parathyroid glands are highly vascularized and display a microRNA (miRNA) profile divergent from normal parathyroid glands (PaNs). Angiogenic miRNAs, namely miR-126-3p, miR-126-5p, and miR-296-5p, have been found downregulated in parathyroid tumors. Here, we show that miR-126-3p expression levels are reduced in parathyroid adenomas (PAds; n = 12) compared with PaNs (n = 4). In situ hybridization (ISH) of miR-126-3p and miR-296-5p in 10 PAds show that miR-126-3p is expressed by endothelial cells lining the walls of great vessels and by cells within the thin stroma surrounding acinar structures. At variance, miR-296-5p was detectable in most PAd epithelial cells. Combining ISH for miR-126-3p with immunohistochemistry for the endothelial and mesenchymal markers CD34, CD31 and α-smooth muscle actin (αSMA), we could identify that miR-126-3p is localized in the αSMA-positive thin stroma. Further, miR-126-3p-expressing cells are enriched in the CD34-positive stromal cells surrounding epithelial cell acinar structures, a cellular pattern consistent with tumor-associated myofibroblasts (TAMs). In line with this, CD34-positive cells, sorted by FACS from PAds tissues, express miR-126-3p at higher levels than CD34-negative cells, suggesting that miR-126-3p downregulation promotes the endothelial-to-αSMA+ mesenchymal transition. In human mesenchymal stem cells derived from bone marrow (hBM-MSCs), a model of TAMs, the co-culture with PAds-derived cells for 5 days decreases miR-126-3p, while it increases VEGFA expression. At variance, adrenomedullin (ADM) expression is unaffected. Finally, overexpression of the miR-126-3p mimic in both hBM-MSCs and PAds-derived explants downregulates VEGFA expression levels. In conclusion, miR-126-3p is expressed by both endothelial cells and TAMs in PAds, and its downregulation promotes neoangiogenesis, possibly through VEGFA overexpression.

Intraoperative angiography with indocyanine green to assess anastomosis perfusion in patients undergoing laparoscopic colorectal resection: results of a multicenter randomized controlled trial.

BACKGROUND: Insufficient vascular supply is one of the main causes of anastomotic leak in colorectal surgery. Intraoperative indocyanine-green (ICG) angiography has been shown to provide information on tissue perfusion, identifying a well-perfused location for colonic and rectal transections, and thus possibly reducing the leak rate. Aim of this study was to evaluate the usefulness of intraoperative assessment of anastomotic perfusion using ICG angiography in patients undergoing left-sided colon or rectal resection with colorectal anastomosis. METHODS: This randomized trial involved 252 patients undergoing laparoscopic left-sided colon and rectal resection randomized 1:1 to intraoperative ICG or to subjective visual evaluation of the bowel perfusion without ICG. The primary aim was to assess whether ICG angiography could lead to a reduction in anastomotic leak rate. Secondary outcomes were possible changes in the surgical strategy and postoperative morbidity. RESULTS: After randomization, 12 patients were excluded. Accordingly, 240 patients were included in the analysis; 118 were in the study group, and 122 in the control group. ICG angiography showed insufficient perfusion of the colic stump, which led to extended bowel resection in 13 cases (11%). An anastomotic leak developed in 11 patients (9%) in the control group and in 6 patients (5%) in the study group (p = n.s.). CONCLUSIONS: Intraoperative ICG fluorescent angiography can effectively assess vascularization of the colic stump and anastomosis in patients undergoing colorectal resection. This method led to further proximal bowel resection in 13 cases, however, there was no statistically significant reduction of anastomotic leak rate in the ICG arm. CLINICAL TRIAL: ClinicalTrials.gov NCT02662946.

Polyphenolic Fraction from Olive Mill Wastewater: Scale-Up and in Vitro Studies for Ophthalmic Nutraceutical Applications.

The valorization of food wastes is a challenging opportunity for a green, sustainable, and competitive development of industry. Approximately 30 million m3 of olive mill wastewater (OMWW) are produced annually in the world as a by-product of the olive oil extraction process. In addition to being a serious environmental and economic issue because of their polluting load, OMWW can also represent a precious resource of high-added-value molecules such as polyphenols that show acclaimed antioxidant and anti-inflammatory activities and can find useful applications in the pharmaceutical industry. In particular, the possibility to develop novel nutraceutical ophthalmic formulations containing free radical scavengers would represent an important therapeutic opportunity for all inflammatory diseases of the ocular surface. In this work, different adsorbents were tested to selectively recover a fraction that is rich in polyphenols from OMWW. Afterward, cytotoxicity and antioxidant/anti-inflammatory activities of polyphenolic fraction were evaluated through in vitro tests. Our results showed that the fraction (0.01%) had no toxic effects and was able to protect cells against oxidant and inflammatory stimulus, reducing reactive oxygen species and TNF-α levels. Finally, a novel stable ophthalmic hydrogel containing a polyphenolic fraction (0.01%) was formulated and the technical and economic feasibility of the process at a pre-industrial level was investigated.