AGO2 localizes to cytokinetic protrusions in a p38-dependent manner and is needed for accurate cell division.

Argonaute 2 (AGO2) is an indispensable component of the RNA-induced silencing complex, operating at the translational or posttranscriptional level. It is compartmentalized into structures such as GW- and P-bodies, stress granules and adherens junctions as well as the midbody. Here we show using immunofluorescence, image and bioinformatic analysis and cytogenetics that AGO2 also resides in membrane protrusions such as open- and close-ended tubes. The latter are cytokinetic bridges where AGO2 colocalizes at the midbody arms with cytoskeletal components such as α-Τubulin and Aurora B, and various kinases. AGO2, phosphorylated on serine 387, is located together with Dicer at the midbody ring in a manner dependent on p38 MAPK activity. We further show that AGO2 is stress sensitive and important to ensure the proper chromosome segregation and cytokinetic fidelity. We suggest that AGO2 is part of a regulatory mechanism triggered by cytokinetic stress to generate the appropriate micro-environment for local transcript homeostasis.

Discovery and Pharmacological Evaluation of STEAP4 as a Novel Target for HER2 Overexpressing Breast Cancer.

Breast cancer (BC) is a highly heterogeneous disease encompassing multiple subtypes with different molecular and histopathological features, disease prognosis, and therapeutic responses. Among these, the Triple Negative BC form (TNBC) is an aggressive subtype with poor prognosis and therapeutic outcome. With respect to HER2 overexpressing BC, although advanced targeted therapies have improved the survival of patients, disease relapse and metastasis remains a challenge for therapeutic efficacy. In this study the aim was to identify key membrane-associated proteins which are overexpressed in these aggressive BC subtypes and can serve as potential biomarkers or drug targets. We leveraged on the development of a membrane enrichment protocol in combination with the global profiling GeLC-MS/MS technique, and compared the proteomic profiles of a HER2 overexpressing (HCC-1954) and a TNBC (MDA-MB-231) cell line with that of a benign control breast cell line (MCF-10A). An average of 2300 proteins were identified from each cell line, of which approximately 600 were membrane-associated proteins. Our global proteomic methodology in tandem with invigoration by Western blot and Immunofluorescence analysis, readily detected several previously-established BC receptors like HER2 and EPHA2, but importantly STEAP4 and CD97 emerged as novel potential candidate markers. This is the first time that the mitochondrial iron reductase STEAP4 protein up-regulation is linked to BC (HER2+ subtype), while for CD97, its role in BC has been previously described, but never before by a global proteomic technology in TNBC. STEAP4 was selected for further detailed evaluation by the employment of Immunohistochemical analysis of BC xenografts and clinical tissue microarray studies. Results showed that STEAP4 expression was evident only in malignant breast tissues whereas all the benign breast cases had no detectable levels. A functional role of STEAP4 intervention was established in HER2 overexpressing BC by pharmacological studies, where blockage of the STEAP4 pathway with an iron chelator (Deferiprone) in combination with the HER2 inhibitor Lapatinib led to a significant reduction in cell growth in vitro. Furthermore, siRNA mediated knockdown of STEAP4 also suppressed cell proliferation and enhanced the inhibition of Lapatinib in HER2 overexpressing BC, confirming its potential oncogenic role in BC. In conclusion, STEAP4 may represent a novel BC related biomarker and a potential pharmacological target for the treatment of HER2 overexpressing BC.

Excessive Leukocytosis Leading to a Diagnosis of Aggressive Thyroid Anaplastic Carcinoma: A Case Report and Relevant Review.

INTRODUCTION: Leukocytosis and particularly neutrophilia are usually caused by acute infection, inflammation, and myeloproliferative neoplasms. However, leukocytosis can also occur in patients with malignancy either due to bone marrow metastases or in the context of a paraneoplastic syndrome. CASE PRESENTATION: An 86-year-old female was admitted to our hospital due to marked leukocytosis (white blood cells [WBC] >40,000/µL), neutrophilia, and monocytosis. She was afebrile and reported hoarseness and mild difficulty swallowing. Upon physical examination, lung auscultation revealed inspiratory wheezing and a non-tender mass was observed in the anterior midline of the neck. Blasts and immature WBC were not found, and polymerase chain reaction for the detection of BCR/ABL gene was negative. A mass (5.4 cm in diameter) of abnormal parenchymal composition with calcifications occupying the right lobe, was seen on thyroid ultrasound. Cytology, after fine-needle aspiration, showed an anaplastic thyroid carcinoma (ATC). The cervical and chest computed tomography scan revealed a low-density lesion with calcifications that shifts and presses the trachea and multiple lung nodular lesions bilaterally. Since the case was inoperable and the airway was severely obstructed, a DUMON stent was placed. Biopsy of specimens from the trachea lesion revealed a tumor with significant atypical cells and focal squamoid features. The patient's WBC increased to 72,470/µL. Additionally, interleukin-6 (IL-6) was markedly elevated (20.2 pg/mL). The patient passed away due to respiratory arrest 55 days after her initial admission. DISCUSSION: Excessive leukocytosis in a patient, having excluded infectious disease and myelodysplastic syndrome, could represent a manifestation of a paraneoplastic syndrome due to various cytokines secretion from the tumor. In our case, ATC synthesized and secreted IL-6, which seems to be the cause of severe leukocytosis.

Reactive C cell hyperplasia as an incidental finding after thyroidectomy for papillary carcinoma.

The biologic and clinical significance of reactive C cell hyperplasia (CCH), adjacent to differentiated thyroid cancers, remains unknown. Our aim was to investigate the presence of CCH in thyroidectomy specimens with papillary thyroid carcinomas (PTC) and discuss its epidemiology and histology. In total, 413 patients were prospectively included in the study (189 benign goiters, 224 PTC). Reactive CCH was observed in 9.8% of PTC cases (32% males, 68% females, mean age 48.3 ± 16.4 years) and usually ipsilateral to the primary tumor (91%). Histologically, CCH was either focal (91%) or diffuse (9%) and almost always (92%) found in the middle or upper thirds of the thyroid lobes. Patients with PTC/CCH were generally younger than patients with benign goiters (0.027). On the other hand, patients with PTC and with PTC/CCH did not differ in terms of age, gender, basal calcitonin levels, primary tumor size, multifocality, extrathyroidal invasion, or lymph node metastasis. Thyroiditis, however, was more frequent in cases with PTC/CCH compared to PTC alone. Reactive CCH is considered a physiological response of the C cells to various stimuli, differentiated thyroid cancer among others. It bears no malignant potential and requires no additional treatment, following thyroidectomy.

IGF-IEc Expression Is Associated With Advanced Differentiated Thyroid Cancer.

BACKGROUND/AIM: Recent knowledge implicates a differential expression of the insulin-like growth factor-I (IGF-I) mRNA splice variants (i.e., IGF-IEa, IGF-IEb and IGF-IEc) in cancerous tissues, implying possible specific roles of the encoded IGF-I protein isoforms in cancer biology. In particular, there is growing evidence that the IGF-IEc isoform may play a distinct biological role in various types of cancers. The present study investigated whether IGF-IEc expression is associated with a particular type of thyroid cancer. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded tissue specimens of different types of thyroid cancers from 92 patients were assessed for IGF-IEc expression by immunohistochemistry. In addition, thyroid cancer biopsies of different TNM staging histological types were evaluated for mRNA expression of the IGF-IEc transcript by real-time polymerase chain reaction (PCR). RESULTS: From the total number of 92 samples, 2 were anaplastic, 10 medullary, 4 hyperplasias of C-cells, 11 follicular, 5 hurtle cell carcinomas, 2 poorly differentiated, 5 nodular hyperplasias, 1 lymphoma and 52 were papillary thyroid cancers. The age of cancer diagnosis or tumor size did not significantly affect the IGF-IEc expression. Among all types of cancers, IGF-IEc was expressed in papillary differentiated thyroid cancer. Its expression/localization was mainly cytoplasmic and significantly associated with TNM staging and the presence of muscular and capsule cancerous invasion (p<0.05). Similarly, a differential profile was revealed regarding the mRNA expression of the IGF-IEc transcript, that exhibited a higher expression in aggressive compared to the non-aggressive papillary cancers. CONCLUSION: IGF-IEc isoform expression in thyroid cancer is positively associated with more advanced stages of papillary thyroid cancer.

PI3K/AKT pathway genetic alterations and dysregulation of expression in bladder cancer.

PURPOSE: To examine the involvement of specific components of the PI3K/AKT pathway in urinary bladder cancer development. METHODS: Samples from 65 tumors and 13 normal bladder tissues were collected. Genomic DNA isolation from snap-frozen and paraffin-embedded laser-microdissected tissues was followed by Sanger sequencing, whereas total RNA was purified for use in RT-PCR analyses. Immunohistochemistry was carried out on sections of paraffin-embedded biopsy material. RESULTS: Three pathogenic mutations (two missense and one frameshift) were identified in exon 20 of PIK3CA {c.3140A>G (p.His1047Arg), c.[3172A>T(;)3174C>T] (p.lle1058Phe), c.3203dupA (p.Asn1068Lysfs*5)} after laser capture microdissection, whereas PTEN mRNA expression was found to be downregulated in bladder cancer tissues compared to normal bladder urothelium. Upregulation of cytoplasmic and nuclear p-AKT expression was detected in low grade tumors, whereas in infiltrating carcinomas p-AKT was shown to be downregulated and confined to the cytoplasm. PTEN expression was weak and mainly cytoplasmic in superficial tumors, but stronger and nuclear in the infiltrating tumors. CONCLUSIONS: PI3K/AKT pathway activation is crucial for bladder cancer initiation and progression. In this context, PIK3CA, p-AKT and nuclear PTEN could be used along with other biomarkers for prognosis and selection of appropriate therapy in the clinical management of bladder cancer.

Resistance to Fas-Mediated Apoptosis Does Not Correlate to Structural Alterations or Expression Changes of the Death Receptor in Papillary Thyroid Carcinomas.

BACKGROUND: Malignant cells exhibit significant resistance to FAS-mediated cell death, through different processes, including FAS mutations, soluble FAS expression, or FAS transcriptional dysregulation by P53, eventually escaping from immune surveillance. Since thyroid carcinomas were shown to be resistant to FAS-mediated apoptosis, we investigated the above mechanisms in thyroid carcinoma samples. METHODS: Thirty-seven thyroid carcinoma samples were analyzed for mutations in FAS exon 9 and TP53 exons 5-8 and protein expression by means of immunohistochemistry. Moreover, thyroid carcinoma mRNA samples were subjected to reverse transcription - PCR, to evaluate the relative expression of transmembrane FAS versus its soluble form. RESULTS: Analysis revealed indications for TP53 mutations in the anaplastic carcinomas, but not in the other thyroid specimens examined for TP53 or FAS exon 9 mutations. FAS receptor expression was observed in almost all thyroid specimens (97%) with significant up-regulation in papillary carcinomas. P53 nuclear staining was observed only in anaplastic carcinomas. Full-length FAS mRNA was detected in all specimens examined, with soluble FAS mRNA being either absent or present in very low amounts. CONCLUSIONS: Our results denote that FAS death domain or TP53 DNA-binding domain mutations, down-regulation of FAS receptor expression, or expression of FAS soluble isoform are not responsible for the seeming inhibition of FAS-mediated apoptosis in papillary thyroid carcinoma cells.

Association of Baseline Neutrophil-to-Lymphocyte Ratio with Clinicopathological Characteristics of Papillary Thyroid Carcinoma.

OBJECTIVE: To investigate the potential association of neutrophil-to-lymphocyte ratio (NLR), a surrogate systemic inflammatory biomarker, with clinical and pathological characteristics of papillary thyroid cancers. METHODS: 205 patients with papillary carcinoma were identified from the institutional thyroid cancer database between 2006 and 2015 (55 males, 150 females, mean age 51.2 ± 14.7 years). NLR was calculated as the absolute neutrophil count divided by the absolute lymphocyte count, based on the preoperative complete blood cell counts. RESULTS: NLR was significantly higher in carcinomas with extrathyroidal invasion (2.74 ± 01.24 versus 2.39 ± 0.96, p = 0.04) and bilateral (2.67 ± 1.15 versus 2.35 ± 0.96, p = 0.03) and multifocal tumours (2.65 ± 1.08 versus 2.29 ± 0.96, p = 0.01), as well as lymph node-positive tumours (3.12 ± 1.07 versus 2.41 ± 1.02, p = 0.03). On the other hand, NLR values were not associated with gender, age, tumour size, histologic subtype, the presence of thyroiditis, and TNM staging. CONCLUSIONS: As an index of inflammation, NLR is inexpensive, readily available, and easy to extract from routine blood tests. We found increased NLR values in papillary carcinomas with poorer histopathological profile and more aggressive clinical behaviour. Whether this systemic inflammatory response, as expressed by the NLR, represents the inflammatory microenvironment leading to tumourigenesis, or is a tumour-associated phenomenon, remains to be elucidated and warrants further study.

Evidence for APOBEC3B mRNA and protein expression in oral squamous cell carcinomas.

It has been demonstrated that APOBEC3B possesses cytidine deaminase activity, which is likely to result in C-to-T signature mutations. Increased expression of the APOBEC3B gene has been shown to correlate with higher incidence of such mutations in various cancer types, such as breast, bladder, lung, and head and neck carcinomas. In the current study, we used in silico methods, immunohistochemistry and qRT-PCR to detect the presence of APOBEC3B signature mutations and examine the levels and patterns of APOBEC3B expression in oral squamous cell carcinomas (OSCCs). Using the Cancer Genome Atlas (TCGA) database, we have found a high incidence of C-to-T transitions in head and neck squamous cell carcinomas (HNSCCs), of which OSCCs constitute the largest subgroup. Additionally, we compared APOBEC3B expression, at both mRNA and protein level, between OSCCs and non-cancerous samples. APOBEC3B was detected in both groups, but nuclear localization was consistent only in normal oral cells. APOBEC3B mRNA levels were clearly higher in OSCCs than in controls. These results suggest that while in normal oral cells APOBEC3B has an important nuclear function to fulfill, this activity may be hindered in a subgroup of tumor cells, due to the more prominent localization of the enzyme in the cytoplasm.

Association of Intraoperative Neuromonitoring With Reduced Recurrent Laryngeal Nerve Injury in Patients Undergoing Total Thyroidectomy.

Importance: Injury of the recurrent laryngeal nerve (RLN) is one of the most serious complications of thyroid surgery. Intraoperative neuromonitoring (IONM) has been introduced to verify RLN function integrity and may be a helpful adjunct in nerve dissection. Objective: To determine whether the use of IONM can reduce the incidence of RLN injury in patients undergoing total thyroidectomy. Design, Setting, and Participants: This cohort study included 2556 patients who underwent total thyroidectomy between January 2002 and December 2012 in the Department of Otolaryngology-Head and Neck Surgery of Venizeleio General Hospital, Heraklion, Greece. Patients who had IONM during the procedure (n = 1481) were compared with patients who underwent surgery with nerve visualization alone (n = 1075). All patients underwent indirect laryngoscopy-fiberoptic nasopharyngoscopy both preoperatively and on day 2 after surgery to assess vocal cord motility. Main Outcomes and Measures: Use of IONM and incidence of RLN injury. Results: A total of 2556 patients (2028 women and 528 men [5112 RLNs at risk]; mean [SD] age, 51.35 [14.18] years; age range, 18-89 years) underwent total thyroidectomy. Univariate analysis showed that the use of IONM resulted in a significant reduction in RLN injury incidence (3.3% vs 0.7%) with a relative risk reduction of 2.6% (odds ratio [OR], 5.15; 95% CI, 3.12-8.49; number needed to treat, 19). Multivariate logistic regression showed that no use of IONM was an independent risk factor for RLN injury in patients who underwent total thyroidectomy (adjusted OR [AOR], 5.44; 95% CI, 3.26-9.09). Additional risk factors for RLN injury were operative time (AOR, 12.91; 95% CI, 6.66-25.06), maximum diameter greater than 45 mm of right thyroid lobe (AOR, 4.91; 95% CI, 3.12-8.56) and left thyroid lobe (AOR, 2.24; 95% CI, 1.39-4.32), extrathyroid extension (AOR, 3.26; 95% CI, 1.62-6.59), incidental parathyroidectomy (AOR, 3.30; 95% CI, 2.13-5.09), and tumor size larger than 10 mm (AOR, 3.24; 95% CI, 1.59-6.62). Conclusions and Relevance: Our findings showed that the use of IONM decreased significantly both temporary and permanent RLN injuries. The technology of IONM is safe and reliable, and this technique is an important adjunct in nerve dissection and functional neural integrity. The routine use of IONM reduced pitfalls and provided guidance for our surgeons in difficult cases, reoperations, and high-risk patients.

18-Fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography scan for monitoring the therapeutic response in experimental Staphylococcus aureus foreign-body osteomyelitis.

BACKGROUND: 18-Fluoro-2-deoxy-D-glucose positron emission tomography combined with computed tomography ((18)F-FDG PET/CT) scan is useful for diagnosis of osteoarticular infections. Whether (18)F-FDG PET/CT scanning may be used for therapeutic monitoring is not clear. The objective of this study was to develop (18)F-FDG PET/CT scanning for monitoring therapeutic response to antimicrobials in experimental Staphylococcus aureus osteomyelitis. METHODS: A total of 22 rabbits were studied. In 20 animals, the right tibia was inoculated intraoperatively with S. aureus. Two control animals were inoculated with normal saline. A needle was placed in the tibia as a foreign body. Infection was allowed to develop for 21 days when (18)F-FDG PET/CT was performed, the needle was removed, and bone specimens were cultured to confirm infection. Antimicrobial therapy with daptomycin was initiated in all successfully infected animals for 1, 3, or 6 weeks. Following completion of treatment, a second (18)F-FDG PET/CT was performed, animals were euthanized, and infected tibias were harvested for quantitative cultures and histology. A positive scan was defined as (18)F-FDG signal activity greater in the infected tibia than that of the contralateral non-infected control tibia. Therapeutic response was measured by the change of (18)F-FDG signal activity in the infected tibia. RESULTS: All successfully infected animals (n = 14), with microbiologically and/or histologically confirmed osteomyelitis, had positive (18)F-FDG PET/CT scans, while the two control animals had negative scans despite the presence of the foreign body [mean maximum standardized uptake value (SUVmax) (±SD) values 2.96 (±0.80) vs. 1 (±1.10), respectively, P = 0.04]. In the 14 successfully infected animals, the mean SUVmax was significantly higher in the infected compared to the uninfected tibia (P < 0.0001). A SUVmax of 1.4, when used as a cutoff for infection, yielded a diagnostic accuracy of 93 %. At the end of treatment, successfully treated animals and saline controls had a negative (18)F-FDG PET/CT scan (n = 4), while animals with persistent infection despite treatment (n = 12) had a positive (18)F-FDG PET/CT scan (SUVmax 1.0-3.0) (p < 0.001). SUVmax values were significantly reduced after 42 days of treatment from 3.15 ± 0.5 (day 7) to 1.71 ± 0.37 (day 42) (p = 0.05). CONCLUSIONS: (18)F-FDG PET/CT scan is a sensitive and specific tool in therapeutic monitoring of experimental foreign-body osteomyelitis.

SIN1, a critical component of the mTOR-Rictor complex, is overexpressed and associated with AKT activation in medullary and aggressive papillary thyroid carcinomas.

BACKGROUND: Mammalian target of rapamycin (mTOR) forms 2 active complexes in the cell: the rapamycin-sensitive mTOR-Raptor (mTORC1) and the rapamycin-insensitive mTOR-Rictor (mTORC2). The latter activates AKT kinase, which promotes tumor cell survival and proliferation by multiple downstream targets. Mammalian stress-activated protein kinase interacting protein 1 (SIN1), an essential subunit of the mTORC2 complex, maintains the integrity of the complex and substrate specificity and regulates Akt activation. The role of mTOR-Rictor complex activation in thyroid carcinogenesis remains unknown. Therefore, we investigated expression patterns of Sin1 in the cells lines of thyroid carcinoma and tumors and their association with AKT activation, histologic type, and tumor aggressiveness. METHODS: Tissue specimens from 42 patients with thyroid cancer, including follicular (5), papillary (18), medullary (16), and poorly differentiated (3) carcinomas were analyzed via immunohistochemistry for SIN1 expression and AKT phosphorylation at Ser473 residue (Ser473-p-AKT). Eight of 18 papillary carcinomas were aggressive histologic variants. In addition, expression of Sin1 and activation of AKT kinase were analyzed in fresh-frozen tissue samples (normal/tumor), primary cell cultures (papillary thyroid carcinoma [PTC]), and an established thyroid cancer cell line (medullary thyroid carcinoma) by Western blotting. RESULTS: With immunohistochemistry, we found that Sin1 was overexpressed in medullary thyroid carcinomas and aggressive variants of papillary thyroid carcinoma compared with conventional papillary and follicular carcinomas (P < .001). Sin1 expression correlated with AKT activation in the entire study group (P = .002). Using Western blot analysis, we found that Sin1 and p-AKT were detected at a greater level in cultured primary cells from aggressive PTC compared with conventional PTC as well as in cell lines of medullary and anaplastic thyroid carcinoma. High expression levels of SIN1 were detected in papillary thyroid carcinomas compared with benign nodules in immunoblots in which we used fresh-frozen patient samples. Two of the Sin1 protein isoforms, p76 and p55, were detected predominantly in PTC samples. CONCLUSION: Sin1, a critical factor of the mTORC2 complex is overexpressed in clinically aggressive thyroid cancer types and is associated strongly with activation of AKT kinase. Sin1-dependent AKT activation might represent a target for experimental therapy.

Bilateral versus unilateral papillary thyroid microcarcinoma: predictive factors and associated histopathological findings following total thyroidectomy.

OBJECTIVE: The extent of thyroidectomy for papillary thyroid microcarcinoma (PTMC) is debatable. This study investigated the rate and predictive factors of bilateral versus unilateral PTMC with the objective of identifying those patients who may benefit from total thyroidectomy. DESIGN: Between January 2001 and December 2008, 2019 patients who underwent total thyroidectomy were examined. A total of 319 patients diagnosed histopathologically as PTMC were included in the study. The predictive value of age at diagnosis, gender, tumor size, multifocality, lymph node metastasis, thyroid capsule invasion and nonincidental diagnosis using univariate and multivariate analyses were retrospectively analyzed. RESULTS: Of the 319 patients with PTMC, 77 (24.1%) presented bilateral disease. In univariate analysis, size of tumor ≥5 mm (p<0.001), multifocality (p<0.001), lymph node metastases (p<0.001), thyroid capsule invasion (p<0.001) and nonincidental diagnosis (p=0.002) were significantly associated with bilaterality. In multivariate analysis, tumor size (p<0.001), multifocality of the primary tumor in the unilateral lobe (p<0.001) and lymph node metastasis (p<0.001) were independent predictive factors for bilateral PTMC. CONCLUSIONS: Tumor size ≥5 mm and multifocality of the primary carcinoma in the unilateral lobe were independent risk factors for bilateral PTMC. Total thyroidectomy should be considered for these patients, which is of importance for the prediction of possible recurrence of disease.

Activation of mTOR signaling in medullary and aggressive papillary thyroid carcinomas.

BACKGROUND: Because mammalian target of rapamycin (mTOR) may be involved in thyroid carcinogenesis, we investigated the expression and activation patterns of mTOR signaling proteins in thyroid carcinoma cells and tumors and their association with tumor histology and aggressiveness. METHODS: Tissue specimens from 50 patients with thyroid cancer were analyzed for eIF4E, a critical downstream target of the mTOR pathway, using immunohistochemistry. In addition, fresh-frozen samples from patients, and primary tumor cell cultures were analyzed for expression and activation of mTOR signaling proteins by Western blot. Moreover, pharmacologic studies with rapamycin were performed. RESULTS: High expression of eIF4E was observed in medullary thyroid carcinomas (MTC) and in aggressive variants of papillary thyroid carcinomas (PTC) as compared with conventional PTC and follicular thyroid carcinomas (P < .0001). The level of eIF4E expression also correlated with tumor stage (P = .002). Using Western blot analysis, p-rpS6, p-4EBP1, 4EBP1, and eIF4E were detected at higher levels in aggressive PTC and MTC cells. Treatment of MTC cells with increasing concentrations of rapamycin resulted in significant cell death and in decreased cell growth associated with deactivation of the mTOR pathway. CONCLUSION: mTOR signaling, which controls protein synthesis through regulation of translation initiation, is activated in aggressive PTC and MTC and represents a promising target for investigational therapies in these patients.

Direct role of adiponectin and adiponectin receptors in endometrial cancer: in vitro and ex vivo studies in humans.

Low adiponectin levels are an independent risk factor for and mediate the effect of obesity on endometrial cancer in epidemiology studies. The direct or indirect mechanisms underlying these findings remain to be elucidated. We first examined the expression of adiponectin receptor 1 (AdipoR1) and 2 (AdipoR2) in normal human endometrium and in endometrial cancer tissues ex vivo. We then used KLE and RL95-2 human endometrial cancer cell lines in vitro to study relative expression of AdipoRs, to investigate the effect of adiponectin on activating intracellular signaling pathways, and to assess its potential to alter malignant properties. We report for the first time that the relative expression level of AdipoR1 is higher than AdipoR2 in human endometrial cancer tissue, but the expression of AdipoRs is not statistically different from nonneoplastic tissues. We also show for the first time in endometrial cancer cell lines in vitro that adiponectin suppresses endometrial cancer proliferation acting through AdipoRs. Adiponectin also increases the expression of the adaptor molecule LKB1, which is required for adiponectin-mediated activation of AMPK/S6 axis and modulation of cell proliferation, colony formation, adhesion, and invasion of KLE and RL95-2 cell lines. These novel mechanistic studies provide for the first time in vitro and ex vivo evidence for a causal role of adiponectin in endometrial cancer.

A decision support system for assisting fine needle aspiration diagnosis of thyroid malignancy.

OBJECTIVE: To assist diagnosis of thyroid malignancy, implementing a decision support system (DSS) using fine needle aspiration biopsy (FNAB) data. STUDY DESIGN: The set of 2,035 thyroid smears contained 1,886 smears of nonmalignancy (class 1) and 150 smears of malignancy (class 2) verified histologically. For each smear, 67 medical features were considered by the expert, forming 2,036 feature vectors, which were fed into a DSS for discriminating between malignant and nonmalignant smears. The DSS comprised a feature selection and classification module using a combination of three classifiers, the artificial neural network, the support vector machines, and the k-nearest neighbor, under the majority vote procedure. RESULTS: The overall classification accuracy of the DSS was 98.6%, marginally better than the FNAB (97.3%). The DSS had lower sensitivity (89.1%) and better specificity (99.4%) compared to the FNAB. Regarding the smears characterized as "suspicious" by FNAB, a significant improvement of overall accuracy was obtained by the proposed DSS system (84.6%) compared to the FNAB (50.0%). CONCLUSION: The proposed DSS provides significant improvement compared to FNAB regarding discrimination of smears characterized by an expert as "suspicious," reducing the number of patients undergoing surgical procedures.

Circulating adiponectin is inversely associated with risk of thyroid cancer: in vivo and in vitro studies.

CONTEXT: Circulating adiponectin has been inversely associated with risk for several malignancies. Its association with thyroid cancer has not yet been evaluated. OBJECTIVE/METHODS: We measured circulating adiponectin levels in 175 thyroid carcinoma patients and 107 controls. We also examined the expression of adiponectin receptors (AdipoR1 and AdipoR2) using immunohistochemistry in 82 thyroid carcinoma tissues and using RT-qPCR in 40 human thyroid carcinoma tissues (32 papillary, six follicular/Hurthle, one anaplastic, one medullary), four normal human thyroid tissue specimens, and the BHP7 and SW579 thyroid cancer cell lines. We then utilized these thyroid cancer cell lines to investigate whether adiponectin could directly regulate cell cycle or apoptosis. RESULTS: Thyroid cancer patients had lower circulating adiponectin levels than controls (17.00 ± 6.32 vs. 19.26 ± 6.28 µg/ml; P < 0.001). Subjects in the highest tertile of circulating adiponectin concentrations had significantly lower odds of developing any type of thyroid carcinoma (odds ratio = 0.29; 95% confidence interval, 0.16-0.55), or papillary thyroid carcinoma (odds ratio = 0.27; 95% confidence interval, 0.14-0.55), before and after adjustment for potential confounders. Both thyroid carcinoma cell lines and tissues expressed AdipoR1 and AdipoR2. Recombinant adiponectin did not exert a clinically significant direct effect on cell cycle, proliferation, or apoptosis in thyroid cancer cell lines in vitro. CONCLUSIONS: Circulating adiponectin is independently and inversely associated with the risk of thyroid cancer. Human thyroid carcinomas and cell lines express adiponectin receptors. However, in the absence of a major direct effect of adiponectin on thyroid cancer cell lines in vitro, the negative association observed herein may be attributed to the metabolic effects of adiponectin.

Serum adiponectin levels and tissue expression of adiponectin receptors are associated with risk, stage, and grade of colorectal cancer.

Adiponectin has been associated with colorectal cancer (CRC) risk. This study aims to investigate the association of both adiponectin and tissue expression of its receptors with CRC risk as well as clinicopathological characteristics, notably stage and grade. Determination of serum adiponectin and immunohistochemical expression of adiponectin receptors in adenocarcinoma/normal colorectal tissue was performed in samples from 104 newly diagnosed CRC patients and 208 age- and sex-matched controls. Multiple logistic regression odds ratios and 95% confidence intervals for CRC risk were derived, controlling for a series of covariates. Serum adiponectin was negatively associated with CRC risk (odds ratio, 0.72; confidence interval, 0.53-0.99) and also with tumor grade (P = .05). Expression of both adiponectin receptors was stronger in adenocarcinoma vs normal tissue (P = .001). AdipoR1 expression was negatively associated with nodal stage (P = .03); AdipoR2 expression was positively associated with tumor, node, metastasis stage (P = .01). Established positive associations with red meat consumption and diabetes, and negative associations with physical exercise and plant food consumption were confirmed along with a more than 60% higher risk associated with central obesity. Adiponectin levels and tissue expression of hormonal receptors seem to be associated not only with CRC risk but also with components of clinicopathological characteristics; given power limitations, these results should be interpreted with caution. The exact nature of the association and the underlying pathophysiological mechanisms need to be further examined in large prospective studies assessing adiponectin and its receptors as novel targets for exploring CRC growth.

Expression of receptor activator of NF-κB ligand and osteoprotegerin in peripheral giant cell granulomas of the jaws.

BACKGROUND: Peripheral giant cell granuloma is a tumor of the jaw characterized by the presence of multinucleated giant cells and mononuclear cells within a fibrous stroma. These lesions are considered to be of a reactive nature rather than neoplastic. Although peripheral giant cell granulomas is a well-described clinical entity, little is known on its pathogenesis. The aim of this study was to investigate the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) expression and immunolocalization in giant cell granulomas. METHODS: RANKL and OPG protein expression was evaluated in 22 peripheral giant cell granulomas samples, by means of immunohistochemistry. Staining was evaluated semi-quantitatively, according to the extent and intensity of the stain. RESULTS: RANKL was expressed in all cases with a cytoplasmic staining pattern, whereas OPG expression was detected in 21 of the 22 cases examined. Active multinucleated giant cells exhibited intense immunoreactivity for both proteins. CONCLUSION: RANKL and OPG are expressed in peripheral giant cell granulomas of the jaw in a manner supporting the osteoclastic nature of giant cells whereas the possible osteoclastic lineage of stromal monocytes remains ambiguous.

Possible association of CEA expression with oxyphilic change but not with C-cell hyperplasia in Hashimoto's thyroiditis.

Reactive C-cell hyperplasia (CCH) has been observed in cases of autoimmune Hashimoto's thyroiditis; however, its occurrence in Graves' disease, the other major autoimmune disorder, has not yet been investigated. On the other hand, although Carcinoembryonic Antigen (CEA) serum levels have been reported elevated in patients with autoimmune thyroid disease (ATD), the source of CEA production at the cellular level is not elucidated. The aim of this study was to evaluate CCH and CEA immunohistochemical expression and comparatively analyze them in 136 ATD cases (107 Hashimoto's and 29 Graves' disease cases) and 20 cases of nodular hyperplasia (NH). Immunohistochemistry using monoclonal antibodies to chromogranin and CEA was performed. A scoring system for CCH and semiquantitative evaluation for CEA expression were applied. C-cell hyperplasia was absent in NH cases. In contrast, it was detected in 11% of ATD cases being more frequently observed in Hashimoto's (12.1%) than Graves' disease (6.8%) CCH associated to male sex and older age of Hashimoto's patients. CEA was detected only in ATD cases (33.8%), in C-cells and in follicular cells as well, being more frequently detected in Graves' (44.8%) than Hashimoto's (30.8%) disease. An interesting finding was an emerging possible association of CEA expression with oxyphilic change but not with C-cell hyperplasia in Hashimoto's thyroiditis. No significant correlation was established between CCH and CEA follicular cell expression in neither disease. In conclusion, C-cell hyperplasia and CEA expression may be encountered in the setting of Hashimoto's thyroiditis and Graves' disease.