Role of fine needle aspiration cytology in the diagnosis and management of Warthin’s tumour of the salivary glands

J. M. Viguer, B. Vicandi, J. A. Jiménez‐Heffernan, P. López‐Ferrer, P. González‐Peramato and C. Castillo
Role of fine needle aspiration cytology in the diagnosis and management of Warthin’s tumour of the salivary glands Objective: Local excision surgical procedures and non‐surgical conservative management are considered alternatives to superficial parotidectomy in the treatment and management of Warthin’s tumour (WT). Such therapeutic alternatives demand accurate diagnosis. In order to determine whether fine needle aspiration cytology (FNAC) is capable of rendering such a minimally invasive diagnosis, we evaluated its accuracy and diagnostic parameters in a large series of histologically proven cases of WT. Methods: A cytohistological study of 116 salivary tumours from 110 patients (four WT were bilateral) with a histological or cytological diagnosis of WT. Results: Histology confirmed the cytological diagnosis in 103 of 114 tumours (90.4%). Two tumours were incorrectly diagnosed on cytology as WT. In 11 cases of WT there was an erroneous or non‐representative cytological diagnosis. The sensitivity was 90.4%, and positive predictive value 98.1%. Regarding malignancy, there were three misdiagnoses. One tumour diagnosed as WT was a low‐grade mucoepidermoid carcinoma. Two cases considered ‘suspicious of squamous cell carcinoma’ corresponded to WT. After review, 81.3% of the cases of WT were considered typical and 18.7% non‐typical; all misdiagnoses were in the latter group. Cytological difficulties could be divided into three areas: (i) absence of one or more diagnostic components; (ii) ‘squamoid’ pattern; and (iii) mucinous metaplasia. Degenerated oncocytes were present in 65% of cases. Conclusions: FNAC offers the possibility of a reliable diagnosis of WT. Pathologists must pay attention to the squamous appearance of degenerated oncocytes. Cytology, when coupled with clinical and image findings, may permit conservative tumour management.     

Acinic Cell Tumour: A Metastasis In the Lung Diagnosed By Electron Microscopy of Aspirated Material

A case of acinic cell carcinoma, metastatic to lung is presented. Fine needle aspiration showed a low-grade adenocarcinoma. Electron microscopy of the aspirated material, however, allowed definite preoperative diagnosis of metastatic acinic cell carcinoma. The lesion was successfully resected and diagnosis confirmed on histology. Ten years previously an acinic cell carcinoma of similar histology had been excised from the left parotid region.     

Fine needle aspiration cytology of salivary gland lesions: advantages and pitfalls

Fine needle aspiration cytology (FNAC) was performed on 52 patients with salivary gland lesions. A definitive cytodiagnosis was possible in 45 cases: a sensitivity of 89% and a specificity of 94% was achieved. The pitfalls of FNAC of salivary gland lesions are reflected by the false positive and false negative rates which were 4%. Errors of cytodiagnosis are due to the morphological variability of these tumours which make sampling and interpretation difficult.     

Activation of blood coagulation in cancer: implications for tumour progression

Several studies have suggested a role for blood coagulation proteins in tumour progression. Herein, we discuss (1) the activation of the blood clotting cascade in the tumour microenvironment and its impact on primary tumour growth; (2) the intravascular activation of blood coagulation and its impact on tumour metastasis and cancer-associated thrombosis; and (3) antitumour therapies that target blood-coagulation-associated proteins. Expression levels of the clotting initiator protein TF (tissue factor) have been correlated with tumour cell aggressiveness. Simultaneous TF expression and PS (phosphatidylserine) exposure by tumour cells promote the extravascular activation of blood coagulation. The generation of blood coagulation enzymes in the tumour microenvironment may trigger the activation of PARs (protease-activated receptors). In particular, PAR1 and PAR2 have been associated with many aspects of tumour biology. The procoagulant activity of circulating tumour cells favours metastasis, whereas the release of TF-bearing MVs (microvesicles) into the circulation has been correlated with cancer-associated thrombosis. Given the role of coagulation proteins in tumour progression, it has been proposed that they could be targets for the development of new antitumour therapies.     

Metastasis‐associated gene,mag‐1 improves tumour microenvironmental adaptation and potentiates tumour metastasis

Metastasis is a major cause of death from malignant diseases, and the underlying mechanisms are still largely not known. A detailed probe into the factors which may regulate tumour invasion and metastasis contributes to novel anti‐metastatic therapies. We previously identified a novel metastasis‐associated gene 1 (mag‐1) by means of metastatic phenotype cloning. Then we characterized the gene expression profile of mag‐1 and showed that it promoted cell migration, adhesion and invasion in vitro. Importantly, the disruption of mag‐1 via RNA interference not only inhibited cellular metastatic behaviours but also significantly reduced tumour weight and restrained mouse breast cancer cells to metastasize to lungs in spontaneous metastatic assay in vivo. Furthermore, we proved that mag‐1 integrates dual regulating mechanisms through the stabilization of HIF‐1α and the activation of mTOR signalling pathway. We also found that mag‐1‐induced metastatic promotion could be abrogated by mTOR specific inhibitor, rapamycin. Taken together, the findings identified a direct role that mag‐1 played in metastasis and implicated its function in cellular adaptation to tumour microenvironment.     

Salivary gland of the tick vector of east coast fever. IV. Cell type selectivity and host cell responses to Theileria parva

Responses of cells in the tick salivary gland to parasitism by Theileria parva were studied by electron microscopy. The gland is composed of three distinct types of acini (I, II, III) which together include ten or more different cell types. Of some 30 infected cells observed in the present study, all were E-cells of acinus III. The parasite thus exhibits a high degree of selectivity for acinus and cell type. The glandular cell invaded undergoes massive hypertrophy and accumulates glycogen deposits in its cytoplasm which may serve as an energy source for the growing intracellular parasite. As synthesis of its secretory material declines the product is packaged in progressively smaller secretory granules. The extensive arrays of endoplasmic reticulum are dismantled and eliminated in autophagic vacuoles. Excess secretory granules are also broken down by crinophagy. After 4 days, sporogony is completed and the host cell contains 30,000–50,000 sporozoites in an electron-lucent cytoplasm largely devoid of cytomembranes and secretory granules. Mitochondria are still present and normal in appearance. The loss of basophilia and secretory granules observed heretofore by light microscopy have been attributed to ingestion and destruction of host organelles by the parasite. The pallid appearance of the cytoplasm has been interpreted as a sign of impending degeneration of the host cell. In electron micrographs no ingestion of organelles by the parasite or degenerative changes were found. The host cell clearly remains viable and metabolically active throughout sporogony. The striking changes in its ultrastructure result from active elimination of organelles and inclusions by the host cell itself in response to parasitism.     

Updates on mechanistic insights and targeting of tumour metastasis

Malignant tumours are one of the major diseases that seriously endanger human health. The characteristics of their invasion and metastasis are one of the main causes of death in cancer patients, and these features cannot be separated from the participation of various molecules‐related cells living in the tumour microenvironment and specific structures. Tumour invasion can approximately be divided into several specific steps according to the movement of tumour cells. In each step, there are different actions in the tumour microenvironment that mediate the interactions among substances. Researchers are attempting to clarify every mechanism of the tumour dissemination. However, there is still a long way to the final determination. Here, we review these interactions in tumour invasion and metastasis at the structural, molecular and cellular levels. We also discuss the ongoing studies and the promise of targeting metastasis in tumour therapy.     

Coupled modelling of tumour angiogenesis, tumour growth and blood perfusion

We propose a mathematical modelling system to investigate the dynamic process of tumour cell proliferation, death and tumour angiogenesis by fully coupling the vessel growth, tumour growth and blood perfusion. Tumour growth and angiogenesis are coupled by the chemical microenvironment and the cell-matrix interaction. The haemodynamic calculation is carried out on the updated vasculature. The domains of intravascular, transcapillary and interstitial fluid flow were coupled in the model to provide a comprehensive solution of blood perfusion variables. An estimation of vessel collapse is made according to the wall shear stress criterion to provide feedback on vasculature remodelling. The simulation can show the process of tumour angiogenesis and the spatial distribution of tumour cells for periods of up to 24 days. It can show the major features of tumour and tumour microvasculature during the period such as the formation of a large necrotic core in the tumour centre with few functional vessels passing through, and a well circulated tumour periphery regions in which the microvascular density is high and associated with more aggressive proliferating cells of the growing tumour which are all consistent with physiological observations. The study also demonstrated that the simulation results are not dependent on the initial tumour and networks, which further confirms the application of the coupled model feedback mechanisms. The model enables us to examine the interactions between angiogenesis and tumour growth, and to study the dynamic response of a solid tumour to the changes in the microenvironment. This simulation framework can be a foundation for further applications such as drug delivery and anti-angiogenic therapies.     

Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis

Cancer‐associated fibroblasts (CAFs) interact with tumour cells and promote growth and metastasis. Here, we show that CAF activation is reversible: chronic hypoxia deactivates CAFs, resulting in the loss of contractile force, reduced remodelling of the surrounding extracellular matrix and, ultimately, impaired CAF‐mediated cancer cell invasion. Hypoxia inhibits prolyl hydroxylase domain protein 2 (PHD2), leading to hypoxia‐inducible factor (HIF)‐1α stabilisation, reduced expression of αSMA and periostin, and reduced myosin II activity. Loss of PHD2 in CAFs phenocopies the effects of hypoxia, which can be prevented by simultaneous depletion of HIF‐1α. Treatment with the PHD inhibitor DMOG in an orthotopic breast cancer model significantly decreases spontaneous metastases to the lungs and liver, associated with decreased tumour stiffness and fibroblast activation. PHD2 depletion in CAFs co‐injected with tumour cells similarly prevents CAF‐induced metastasis to lungs and liver. Our data argue that reversion of CAFs towards a less active state is possible and could have important clinical implications.     

Pre-clinical and clinical significance of heparanase in Ewing's sarcoma

Heparanase is an endoglycosidase that specifically cleaves heparan sulphate side chains of heparan sulphate proteoglycans, activity that is strongly implicated in cell migration and invasion associated with tumour metastasis, angiogenesis and inflammation. Heparanase up-regulation was documented in an increasing number of human carcinomas, correlating with reduced post-operative survival rate and enhanced tumour angiogenesis. Expression and significance of heparanase in human sarcomas has not been so far reported. Here, we applied the Ewing's sarcoma cell line TC71 and demonstrated a potent inhibition of cell invasion in vitro and tumour xenograft growth in vivo upon treatment with a specific inhibitor of heparanase enzymatic activity (compound SST0001, non-anticoagulant N-acetylated, glycol split heparin). Next, we examined heparanase expression and cellular localization by immunostaining of a cohort of 69 patients diagnosed with Ewing's sarcoma. Heparanase staining was noted in all patients. Notably, heparanase staining intensity correlated with increased tumour size (P = 0.04) and with patients' age (P = 0.03), two prognostic factors associated with a worse outcome. Our study indicates that heparanase expression is induced in Ewing's sarcoma and associates with poor prognosis. Moreover, it encourages the inclusion of heparanase inhibitors (i.e. SST0001) in newly developed therapeutic modalities directed against Ewing's sarcoma and likely other malignancies.     

Development and characterization of SV40 immortalized rat parotid acinar cell lines

Summary Rat parotid salivary gland acinar cells were transfected by CaPO₄ precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 30 clonal cell lines, 2 were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the β-adrenergic agonist (isoproterenol), vasoactive intestinal peptide prostaglandin E₁, and forskolin were effective activators of intracellular cyclic adenosine 3′:5′-cyclic monophosphate production. Phenylephrine, carbamylcholine, and UTP were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without affect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express the SV40 large T antigen. Electron microscopic evaluation documented moderate to high levels of cytodifferentiation with the maintenance of tripartite junctional complexes, cellular polarization, and presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 22 and 36 h, respectively.     

LINC00473 antagonizes the tumour suppressor miR‐195 to mediate the pathogenesis of Wilms tumour via IKKα

Objectives

Although dramatic improvements of overall survival has achieved in patients with favourable histology Wilms tumour, disease recurrence is still the main cause of cancer‐related death in childhood. Long non‐coding RNAs (lncRNAs) as oncogenes or tumour suppressors are dysregulated during carcinogenesis. However, the role of lncRNAs in the pathogenesis of Wilms tumour is unknown. Here, an lncRNA LINC00473 signature that functioned as oncogene was identified in Wilms tumour.

Methods

Wilms tumour (n = 15) and relative normal tissues were collected. The LINC00473 expression and function in Wilms tumour was determined. The LncRNA‐miRNA network of LINC00473 was analysed in vitro and vivo.

Results

We uncovered that the expression of LINC00473 was elevated in tumour tissues than that in relative normal tissues. Higher LINC00473 levels correlated to higher stage and unfavourable histology Wilms tumour. Mechanistically, knockdown of LINC00473 inhibited cell vitality and induced Bcl‐2‐dependent apoptosis and G1/S arrest via CDK2 and cyclin D1. Moreover, LINC00473 harboured binding sites for miR‐195 and limited miR‐195 availability in a dose‐dependent manner. Forced expression of miR‐195 impaired tumour survival and metastasis, which, however, could be restored by LINC00473. Furthermore, IKKα was the downstream of LINC00473/miR‐195 signals and could be directly targeted by miR‐195 to participate LINC00473‐induced tumour progression. Loss‐of‐function of LINC00473 in vivo effectively promoted the regression of Wilms tumour via miR‐195/IKKα‐mediated growth inhibition.

Conclusion

LINC00473 as an oncogene is up‐regulated to participate into the molecular pathogenesis of Wilms tumour via miR‐195/IKKα.     

Human salivary gland branching morphogenesis: morphological localization of claudins and its parallel relation with developmental stages revealed by expression of cytoskeleton and secretion markers

Development of salivary glands is a highly complex and dynamic process termed branching morphogenesis, where branched structures differentiate into mature glands. Tight junctions (TJ) are thought to play critical roles in physiological functions of tubular organs, contributing to cell polarity and preventing lateral movement of membrane proteins. Evidence demonstrated that claudins are directly involved in TJ formation and function. Using immunohistochemistry and immunofluorescence we have mapped the distribution of claudins-1, 2, 3, 4, 5, 7 and 11 and compared it with the expression of differentiation markers in human salivary glands obtained from foetuses ranging from weeks 4 to 24 of gestation. Expression of all claudins, except claudin-2 was detected in the various phases of human salivary gland development, up to fully mature salivary gland. The expression of all claudins increased according to the progression of salivary gland maturation evidenced by the classical markers-cytokeratin 14, cytokeratin low molecular weight, smooth muscle actin and human secretory component. Tight junction proteins-claudins appear to be important in the final shape and physiological functions of human salivary glands and are parallel related with markers of salivary gland differentiation.     

Development and characterization of SV40 immortalized rat submandibular acinar cell lines

Summary Rat submandibular salivary gland acinar cells were transfected by CaPO₄ precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 27 clonal cell lines, two were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the β-adrenergic agonist, isoproterenol, vasoactive intestinal peptide, and prostaglandin E₁ were effective activators of intracellular cyclic AMP production. Epinephrine, norepinephrine, phenylephrine, acetylcholine, and P_2U-purinoceptor agonists were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without effect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express glutamine/glutamic acid-rich proteins, a submandibular acinar cell specific secretory protein family. Electron microscopic evaluation documented the maintenance of tripartite junctional complexes, cellular polarization, and the presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 25 h.     

Effects of oxygen,insulin, and glucagon concentrations on rat submandibular acini in serum-free primary culture

Summary The objective of these studies was to develop serum-free culture conditions for dissociated acini from rat submandibular glands. Acini were isolated from the submandibular glands of 42–46 d old rats and cultured on reconstituted rat tail collagen containing laminin in 1:1 Ham’s F12 and Dulbecco’s media, supplemented with BSA, transferrin, insulin, T₃, EGF, dexamethasone, retinoic acid, carbamylcholine, and trace elements, and gassed with 50% O₂. The acini became partly embedded in the collagen gel and rapidly enlarged throughout the first 22 d of culture, maintaining modest seromucous acinar differentiation, as judged morphologically and by mucin secretion. Parallel cultures then were grown under 20, 35, 50, and 65% O₂, and evaluated morphologically and by DNA content. Growth and retention of seromucous acinar characteristics were best with 35% O₂, but lipid accumulation and cell death were unacceptably high. A spectrum of concentrations of insulin and glucagon then were tried. With 0.05μg/ml insulin, cellular growth and organization were orderly, lipid accumulations were not excessive, and moderate differentiation was retained through 15 d of culture. With more than 0.1μg/ml insulin added to or subtracted from the optimum, the detrimental effects recurred. Addition of sufficient glucagon counteracted the effects of both optimum and excessive concentrations of insulin. We now have achieved an orderly growth of moderately differentiated rat submandibular acini for 15 d in serum-free primary culture.     

Accumulation of unusual sterile transcripts of TCRβ in mouse hybridoma, murine tumour and non-human primate marmoset tumour

Accumulation of immunoglobulin Ig RNA (from several loci viz., C_H, C_α, J_k-C_k and S_μ during Igμ isotype switching) in B cells and T cell receptor (TCR) RNAs (α, β andγ) in T cells of unusual sizes emanating from germline and rearranged genes were reported to accumulate in human and mouse (and murine too). The precise mechanism and function of these sterile RNA species are yet to be delineated. Similar accumulation of RNA species of unusual sizes were identified with DNA-RNA hybridization and isolation of cDNA employing with DNA and antibody probes in mouse hybridoma, murine tumour, non-human primate marmoset tumour and human leukemic cells.     

Diversity patterns and food web structure in a Mediterranean intermittent stream

In this work we present the results of a one‐year study on the macroinvertebrate community in an intermittent stream in southern Spain. We have studied the taxonomic composition, diversity and food web monthly in order to consider temporal variability in these parameters. More than 60 macroinvertebrate taxa have been recorded in the stream, but they do not cohabit at the same time. Many of them join the community at the beginning of the wet period. Afterwards, some new taxa incorporate but some others disappear. This leads to huge fluctuations in the diversity of the community and in the food web of different sampling dates. These variations are linked to environmental disturbances, mainly to flow peaks and minimums. From a relatively well structured, but simple, food web at the beginning of the wet period, it can be seen how several trophic levels disappear with time, and how several organisms change or extend their trophic function within it. We relate these variations to the intermediate disturbance hypothesis and discuss the resilience of the community of this intermittent stream (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)