MicroRNA expression profiling of RAS-mutant thyroid tumors with follicular architecture: microRNA signatures to discriminate benign from malignant lesions

Purpose: RAS mutations represent common driver alterations in thyroid cancer. They can be found in benign, low-risk and malignant thyroid tumors with follicular architecture, which are often diagnosed as indeterminate nodules on preoperative cytology. Therefore, the detection of RAS mutations in preoperative setting has a suboptimal predictive value for malignancy. In this study, we investigated differentially expressed microRNA (miRNA) in benign and malignant thyroid tumors with follicular architecture carrying mutations in RAS genes. Methods: Total RNA was purified from 60 RAS-mutant follicular-patterned thyroid tumors, including follicular adenoma (FA), noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), papillary and follicular thyroid carcinoma cases (PTC, FTC); 22 RAS-negative FAs were used as controls. The expression analysis of 798 miRNAs was performed by digital counting (nCounter nanoString platform). Results: Comparing RAS-mutant and RAS-negative FAs, 12 miRNAs showed significant deregulation, which was likely related to the oncogenic effects of RAS mutations. Twenty-two miRNAs were differentially expressed in RAS-mutant benign versus malignant tumors. Considering the tumor type, 24 miRNAs were deregulated in PTC, 19 in NIFTP, and seven in FTC and compared to FA group; among these, miR-146b-5p, miR-144-3p, and miR-451a showed consistent deregulation in all the comparisons with the highest fold change. Conclusions: The miRNA expression analysis of follicular-patterned thyroid tumors demonstrated that RAS mutations influences miRNA profile in benign tumors. In addition, several miRNAs showed a histotype-specific deregulation and could discriminate between RAS-mutant benign and RAS-mutant malignant thyroid lesions, thus deserving further investigation as potential diagnostic markers

Indeterminate Thyroid Nodules: From Cytology to Molecular Testing

Thyroid cancer is the most common malignancy of the endocrine system. Fine-needle aspiration (FNA) biopsy of thyroid nodules has become the gold standard procedure, in terms of cost and efficacy, for guiding clinicians towards appropriate patients’ management. One challenge for cytopathologists is to accurately classify cytological specimens as benign or malignant based on cytomorphological features. In fact, with a frequency ranging from 10% to 30%, nodules are diagnosed as indeterminate. In recent years, the mutational landscape of thyroid tumors has been extensively described, and two molecular profiles have been identified: RAS-like (NRAS, HRAS, and KRAS mutations; EIF1AX mutations; BRAF K601E mutation; and PPARG and THADA fusions) and BRAFV600E-like (including BRAFV600E mutation and RET and BRAF fusions). The purpose of this review is to discuss the latest molecular findings in the context of indeterminate thyroid nodules, highlighting the role of molecular tests in patients’ management

Expression of interleukin 6 (IL-6) correlates with oestrogen receptor in human breast carcinoma

Multifunctional cytokines play important and only partially defined roles in mammary tumour development and progression. Normal human mammary epithelial cells constitutively produce interleukin 6 (IL-6), IL-8 and a non-secreted form of tumour necrosis factor. Transformation of mammary epithelial cells by different oncogenes is frequently associated with alterations of cytokine/growth factor production and responsiveness. In the present study we analysed the expression of IL-6 in 149 cases of invasive breast carcinoma and the data have been correlated with clinico-pathological variables including tumour size, histological grade, nodal status, and oestrogen and progesterone receptors, Ki67 and p53, protein expression. Though the majority of breast carcinomas expressed at least low levels of immunoreactive IL-6, we found that expression of this cytokine was inversely associated with histological tumour grade (P = 0.0017), but not with tumour size and nodal status. Ki67 positivity was inversely correlated with IL-6 expression (P = 0.027). Among biological parameters analysed, a direct association was found between the percentage of IL-6-positive cells and that of oestrogen (P = 0.00005) and progesterone (P = 0.025) receptor-positive cells. No correlation was observed between IL-6 and p53 protein expression. These data indicate that down regulation of IL-6 is associated with highly malignant mammary carcinomas. It will be of interest to evaluate whether alterations of cytokines that are constitutively produced by mammary cells are also associated with high-grade tumours

Apoptosis and proliferation in thyroid carcinoma: correlation with bcl-2 and p53 protein expression.

The aim of this study was to determine the apoptotic cell death in 92 thyroid carcinomas of different histotypes (42 papillary, PTC; 12 poorly differentiated, PDC: 21 undifferentiated, UC; and 17 medullary, MC) by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labelling (TUNEL). Apoptotic index (Al, evaluated as a percentage of TUNEL-positive cells of neoplastic cells) was calculated in each tumour. The AI was very low in all subtypes of thyroid carcinoma, ranging from a median value of 0.2 in PTC to 1.4 in UC. The proliferative activity was determined by immunohistochemistry using monoclonal antibody, MIB-1. The percentage of proliferating cells was significantly different among the histotypes, increasing with tumour aggressiveness (from the mean value of 3.1 for PTC to 5.6 for PDC and 51.8 for UC). In addition, the ratio between proliferative activity and apoptosis was significantly higher in UC than in the other histotypes. The expression of bcl-2 and p53 protein (important in the modulation of cell death) was correlated (bcl-2, inverse correlation, r2 = 0.1, P = 0.04; p53, direct correlation, r2 = 0.11, P = 0.02) with apoptotic index in PTC

Do female association preferences predict the likelihood of reproduction?

Sexual selection acting on male traits through female mate choice is commonly inferred from female association preferences in dichotomous mate choice experiments. However, there are surprisingly few empirical demonstrations that such association preferences predict the likelihood of females reproducing with a particular male. This information is essential to confirm association preferences as good predictors of mate choice. We used green swordtails (<i>Xiphophorus helleri</i>) to test whether association preferences predict the likelihood of a female reproducing with a male. Females were tested for a preference for long- or short-sworded males in a standard dichotomous choice experiment and then allowed free access to either their preferred or non-preferred male. If females subsequently failed to produce fry, they were provided a second unfamiliar male with similar sword length to the first male. Females were more likely to reproduce with preferred than non-preferred males, but for those that reproduced, neither the status (preferred/non-preferred) nor the sword length (long/short) of the male had an effect on brood size or relative investment in growth by the female. There was no overall preference based on sword length in this study, but male sword length did affect likelihood of reproduction, with females more likely to reproduce with long- than short-sworded males (independent of preference for such males in earlier choice tests). These results suggest that female association preferences are good indicators of female mate choice but that ornament characteristics of the male are also important

Bcl-2 protein: a prognostic factor inversely correlated to p53 in non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) prognosis is strictly related to well-established clinicopathological parameters which have unfortunately become insufficient in the prognostic evaluation of this type of cancer. As p53 and bcl-2 gene deregulations are frequently involved in several types of epithelial malignancies, we investigated the Bcl-2 and p53 protein expression in 91 and 101 cases of NSCLC respectively. The expression was then compared with established indicators of prognosis and biological behaviour of the tumours. No relationship was observed between Bcl-2 and either clinicopathological or biological parameters such as histology, grading, tumour status, nodal metastasis and proliferative activity evaluated by scoring proliferating cell nuclear antigen expression and Ki-67 immunoreactivity. However, the mean Bcl-2 expression was significantly lower in patients who developed metastasis during follow-up or died of metastatic disease (P = 0.006 and P = 0.01 respectively). Moreover, survival probability was higher in patients who expressed the Bcl-2 protein (P = 0.0002). In contrast with this, p53 protein accumulation was observed in tumours with metastatic nodal involvement (P = 0.02) or in patients who developed metastasis during follow-up (P = 0.01), although no correlation was found between p53 expression and overall survival. An inverse relationship was also found between Bcl-2 and the anti-oncogene protein product p53 (P = 0.01). Thus, a high proportion of NSCLCs express p53 and Bcl-2 proteins and their expression may have prognostic importance

Molecular genetic features and risk assessment in a series of 30 patients who underwent an operation for gastrointestinal stromal tumours

Background: The objective of the study was to investigate the relationship between molecular genetic features and the standard criteria of risk assessment in patients affected by gastrointestinal stromal tumours (GISTs). Methods: A review was conducted of a series of 30 patients, with a mean age of 67 years, who underwent surgery for primary GISTs. R0 resection was accomplished in 27 patients. CD117, CD34 desmin, vimentin, S-100 and smooth muscle actin were immunohistochemically tested to achieve a diagnosis of GIST. The loss of wild-type KIT or platelet-derived growth factor receptor alpha (PDGFRα) genes was investigated by sequencing the tumour DNA. Results: Tumour genes mutations were reported in 23 patients (77%), and wild-type in seven. Mutations on the KIT gene occurred in 18 patients, and mutations on the PDGFRα gene in five. The average sizes of the GIST were 8.7 cm, 5.4 cm and 5.9 cm for KIT gene-mutated, PDGFRα gene-mutated and wild-type tumours, respectively. KIT gene mutations were detected in 50% of gastric and in 70% of extragastric GISTs. Moreover, 70% of tumours with a mitotic rate ≥ 5 x 50 highpower fields (HPFs) underwent KIT gene mutations. Conversely, PDGFRα mutations were observed only in gastric GISTs with a mitotic rate ≤ 5 x 50 HPFs. By stratifying GISTs according to classes of risk, KIT mutation was shown in most of the high-risk tumours. PDGFRα mutations occurred exclusively in lower classes of risk. Conclusion: Molecular analysis data might have a role as a prognostic variable in models of risk assessment for patients with GISTs

Tumour necrosis factor- α and transforming growth factor- β are significantly associated with better prognosis in non-small cell lung carcinoma: putative relation with BCL -2-mediated neovascularization

Recent in vivo and in vitro studies have demonstrated a wide spectrum of biologic activities of cytokines in the pathogenesis and progression of malignancy. Tumour necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β) have emerged as two of the many host-derived mediators that seem to interfere with both antiproliferative and tumorigenic effects in malignant tumours including lung cancer. However, their association with tumour prognosis or prognostic factors has not yet been completely clarified. In this study, we assessed TNF-α and TGF-β mRNA expression by RT-PCR technique in 61 NSCLC samples, demonstrating the presence of TNF-α and TGF-β mRNA in 55.74% and 45.9% of cases, respectively. We also evaluated the expression of the two distinct transmembrane TNF receptors. TNFR-I and TNFR-II, with a PCR-positive signal in 70.49% and 65.57% of cases, respectively. In 49 of the 61 cases, we evaluated the prognostic impact of the two growth-inhibiting factors using the Kaplan–Meier analysis. In the univariate analysis patients without nodal metastatic involvement (P = 0.02), less advanced tumour stage (P = 0.02) or TNF-α and TGF-β positive cancers (P = 0.01 and P = 0.03) showed a favourable prognosis in terms of overall survival. Since our previous studies demonstrated a significant association between NSCLC behaviour, neoangiogenesis and bcl -2 expression, we investigated the putative relation between TNF-α and TGF-β on the one hand, and vascular count (as a measure of tumour angiogenesis) and bcl -2 protein expression, on the other hand. Our results showed a significant direct association between TNF-α and bcl -2 (P = 0.05) and an inverse association between TNF-α and microvessel count (P = 0.03). Moreover, as previously demonstrated, we observed a significant inverse correlation between bcl -2 protein expression and vascular count (P = 0.05), suggesting that the favourable effect of TNF-α on clinical outcome may be related to a bcl -2-mediated low neovascular development. © 2000 Cancer Research Campaig

b-Gamma-glutamyltransferase activity in human vulnerable carotid plaques.

Objective: The atherosclerotic plaque that is vulnerable to rupture and to superimposed thrombosis is mainly represented by a thin-cap fibroatheroma with or without ulceration/thrombosis and inflammatory infiltrates. Total serum gamma-glutamyltransferase (GGT) activity is an independent predictor for cardiovascular events. Four GGT fractions have been identified in plasma and only one of them (b-GGT) in atherosclerotic plaques, but the possible role of GGT in plaque pathophysiology has not been assessed yet. We investigated the relationships between plaque b-GGT activity and the histological features of plaque vulnerability. Methods and results: Plaque GGT activity was investigated in 65 patients undergoing carotid endarterectomy; plaques were histologically characterized and immunostained for GGT. Intra-plaque total and fractional GGT activity was determined by a cost-effective test of molecular size exclusion chromatography, and compared with histological markers of plaque vulnerability. Plaque cholesterol content was also measured by chromatography. b-GGT was the only fraction detected within the atherosclerotic plaques and intra-plaque b-GGT activity correlated to plaque cholesterol content (r = 0.667, P < 0.0001), plasma b-GGT and f-GGT fractions (r = 0.249; r ¼ 0.298, both P < 0.05). Higher b- GGT activity was found in thin-cap fibroatheromas and it was associated to histological markers of vulnerable plaques, i.e., larger necrotic areas, greater macrophage infiltration and higher cholesterol content (P < 0.05). Conclusions: intra-plaque b-GGT activity correlates with the histological markers of vulnerable plaque and with plasma b-GGT in human carotid atherosclerosis; these data support the possible role of b-GGT in clinically significant atherosclerotic disease