EGFR and HER2–Akt–mTOR signaling pathways are activated in subgroups of salivary gland carcinomas

Salivary gland carcinomas encompass a wide spectrum of histological entities. To identify candidate therapeutic targets and innovative treatment options for these carcinomas, we examined epidermal growth factor receptor (EGFR), phosphorylated EGFR (p-EGFR), HER2, and phosphorylated forms of Akt (p-Akt) and mammalian target of rapamycin (p-mTOR) in 47 salivary gland tumors using immunohistochemistry. EGFR overexpression was found in 51?% of the tumors (24/47); in particular, EGFR overexpression occurred in mucoepidermoid (seven out of seven) and salivary duct carcinomas (9/12). Although EGFR amplification was not detected by fluorescence in situ hybridization analysis, increased copy number due to polysomy of chromosome 7, which houses EGFR, was observed in 4 of the 24 tumors with EGFR overexpression; this polysomy occurred most frequently in salivary duct carcinomas (three out of nine). HER2 overexpression was observed in 21?% (10/47) of all tumors; in these 10 tumors, HER2 gene amplification was found in seven cases. p-Akt was found in 51?% (24/47) of all tumors, most frequently in mucoepidermoid carcinomas (six out of seven). p-mTOR was found in 57?% of the latter (four out of seven). Consequently, different signaling cascades were found activated: (1) an EGFR/HER2(-Akt)-mTOR-dependent axis, with gene gains of HER2 and/or EGFR, activated in salivary duct carcinoma and carcinoma ex pleomorphic adenoma; (2) an EGFR(-Akt)-mTOR-dependent pathway activated in mucoepidermoid carcinoma or acinic cell carcinoma, without HER2 or EGFR gene alterations; and (3) an Akt-dependent pathway without EGFR/HER2 activation in other types. These findings indicate that phosphoprotein mapping of components in the EGFR/HER2-Akt-mTOR pathways may be a useful guide to select appropriate targeting regimens.     

The preparation of PLL–GRGDS modified PTSG copolymer scaffolds and their effects on manufacturing artificial salivary gland

We prepared two-dimentional (2D) and three-dimentional (3D) scaffolds with biodegradable poly(butylene terephthalate)-co-poly(butylene succinate)-b-poly(ethylene glycol) (i.e. PTSG), mainly for the purpose of investigating its cytocompatibility and mechanical property as artificial salivary gland material. The surface of 2D scaffold (i.e. PTSG film) was modified by O₂ plasma treatment and the following coating of Gly–Arg–Gly–Asp–Ser (GRGDS) decorated poly(L-lysine) (i.e. PLL–GRGDS). The obtained film was named PLL–GRGDS/PTSG (O). Its surface properties were characterized using contact angles, surface energies, X-ray photoelectron spectroscopy and Fourier transform infrared; and cytocompatibility tests in vitro including morphology, attachment and proliferation of human salivary gland (HSG) epithelial cells were further performed on PTSG films. Meanwhile, 3D scaffold with the shape of porous tube was constructed using hydrogel-rapid prototyping and the performance of 3D scaffold including mechanical property, pore structure, degradation and water uptake was also evaluated. Results revealed that PLL–GRGDS/PTSG (O) possessed the high surface free energy (63.89?mJ/m²) and could immobilize a great amount of PLL–GRGDS, which attributed to the formation of some polar oxygen-containing groups such as carboxyl and carbonyl ones in the process of O₂ plasma treatment. Cell tests in vitro suggested the efficiency of surface modification in enhancing the cytocompatibility of PTSG. Furthermore, the manufacturing scaffold was proved to possess excellent pore structures (porosity 88.9%, connectivity 97.5% and average pore size 35.4?μm) and good mechanical properties (E-modulus 88.4?±?4.1?kPa, yield stress 45.7?±?2.3?kPa, yield strain 56?±?2%, fracture stress 52.2?±?3.5?kPa and fracture strain 63?±?3%). After four weeks hydrolysis reaction, the degradation of the scaffold reached 8% and equilibrium water uptake declined from 51 to 45%. The decline of water uptake was probably caused by the decrease of the hydrophilic units in PTSG copolymer during degradation. These results satisfied the demands for constructing the artificial salivary gland scaffold.     

Minor salivary gland biopsy: Its role in the classification and prognosis of Sjögren's syndrome

Sjögren's syndrome (SS) is an autoimmune disorder characterized by mononuclear cell infiltration in the exocrine glands, which leads to sicca syndrome (xerostomia and xerophthalmia). The etiology of SS is unknown, but multiple environmental factors (infectious, hormonal and stress-related), as well as genetic factors, may play a role in its pathogenesis. The diagnosis of SS is complex considering its clinical and paraclinical parameters may not be very specific. The minor salivary gland biopsy (MSGB) has undoubtedly become crucial for classifying and determining the prognosis of SS. The three main different classification systems for its interpretation have been described by Chisholm and Mason, Greenspan and Daniels, and Tarpley. However, this invasive procedure has variable sensitivity and specificity as well as low reproducibility. The use of additional methods, such as skin biopsy, imaging techniques, and serum/salivary biomarkers, may be combined with current methods to develop a bioscore that could increase diagnostic performance. In this review, we summarized the main pathological findings in SS and the prognosis of patients with SS according to the biopsy results.     

Where Birt–Hogg–Dubé meets Cowden Syndrome: mirrored genetic defects in two cases of syndromic oncocytic tumours

Birt–Hogg–Dubè (BHD) is an autosomal dominant syndrome characterised by skin fibrofolliculomas, lung cysts, spontaneous pneumothorax and renal cancer. The association of benign cutaneous lesions and increased cancer risk is also a feature of Cowden Syndrome (CS), an autosomal dominant disease caused by PTEN mutations. BHD and CS patients may develop oncocytomas, rare neoplasias that are phenotypically characterised by a prominent mitochondrial hyperplasia. We here describe the genetic analysis of a parotid and a thyroid oncocytoma, developed by a BHD and a CS patient, respectively. The BHD lesion was shown to maintain the wild-type allele of FLCN, while losing one PTEN allele. On the other hand, a double heterozygosity for the same two genes was found to be the only detectable tumorigenic hit in the CS oncocytoma. Both conditions occurred in a context of high chromosomal stability, as highlighted by comparative genomic hybridisation analysis. We conclude that, similarly to PTEN, FLCN may not always follow the classical Two Hits model of tumorigenesis and may hence belong to a class of non-canonical tumour suppressor genes. We hence introduce a role of PTEN/FLCN double heterozygosity in syndromic oncocytic tumorigenesis, suggesting this to be an alternative determinant to pathogenic mitochondrial DNA mutations, which are instead the genetic hallmark of sporadic oncocytic tumours.     

Effects of 5-aza-2′deoxycytidine on RECK gene expression and tumor invasion in salivary adenoid cystic carcinoma

Reversion-inducing cysteine-rich protein with kazal motifs (RECK), a novel tumor suppressor gene that negatively regulates matrix metalloproteinases (MMPs), is expressed in various normal human tissues but downregulated in several types of human tumors. The molecular mechanism for this downregulation and its biological significance in salivary adenoid cystic carcinoma (SACC) are unclear. In the present study, we investigated the effects of a DNA methyltransferase (DNMT) inhibitor, 5-aza-2′deoxycytidine (5-aza-dC), on the methylation status of the RECK gene and tumor invasion in SACC cell lines. Methylation-specific PCR (MSP), Western blot analysis, and quantitative real-time PCR were used to investigate the methylation status of the RECK gene and expression of RECK mRNA and protein in SACC cell lines. The invasive ability of SACC cells was examined by the Transwell migration assay. Promoter methylation was only found in the ACC-M cell line. Treatment of ACC-M cells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression of mRNA and protein, and 5-aza-dC significantly suppressed ACC-M cell invasive ability. Our findings showed that 5-aza-dC inhibited cancer cell invasion through the reversal of RECK gene hypermethylation, which might be a promising chemotherapy approach in SACC treatment.     

Immunohistochemical analyses of E-cadherin, β-catenin,CD44s,and CD44v6 expressions,and Ki-67 labeling index in intraductal papillary mucinous neoplasms of the pancreas and associated invasive carcinomas

We examined the expressions of adhesion molecules (E-cadherin, β-catenin, CD44s, and CD44v6) and Ki-67 labeling index (Ki-67 LI) in low- and moderate-grade dysplasia and invasive carcinoma components in ten noninvasive intraductal papillary mucinous neoplasms (IPMNs) of the pancreas and eight invasive carcinomas associated with IPMNs of the pancreas using immunohistochemical methods. There was no significant difference in regard to the proportion of components expressing either E-cadherin or β-catenin in more than 70% of the tumor cells between the low- and moderate-grade dysplasia components. In contrast, the proportion of those in invasive carcinoma components was significantly lower than in low- or moderate-grade dysplasia components. Also, there was no significant difference in the proportion of components expressing CD44s or CD44v6 in more than 5% of tumor cells among low-grade dysplasia, moderate-grade dysplasia, and invasive carcinoma components. In contrast, the Ki-67 LI values increased in the order of low-grade dysplasia, moderate-grade dysplasia, and invasive carcinoma components, with significant differences among them. The present results indicate that carcinoma components are associated with a decrease in tumor cells expressing E-cadherin and β-catenin and have the highest proliferative activity.     

Influence of the sample collection method on salivary interleukin–6 levels in resting and post-exercise conditions

Previous studies demonstrated that no significant relationships exist between salivary and serum IL-6 in resting conditions and following exercise and that appropriate saliva collection procedures allow to avoid analytical drawbacks. This investigation aimed to: (a) compare the effects of two methods of saliva collection on IL-6 assay; (b) search for correlation between salivary and serum IL-6 in resting and post-exercise conditions; (c) evaluate the IL-6 response to isometric contractions. Seventeen sedentary subjects and fifteen athletes underwent one blood and two salivary draws: saliva was collected chewing on cotton salivettes and using a plastic straw (SA method and ST method, respectively). Afterwards, the athletes only completed a fatiguing isometric exercise of the knee extensors and blood and saliva were sampled after the exercise. In the entire group (n = 32), ST method produced higher IL-6 levels than SA method and serum sampling. The exercise elicited significant responses of lactate, serum IL-6, salivary IL-6 (by ST method): salivary IL-6 values using the ST collection method were higher at each sampling point than with the SA method. The correlation analyses applied to both resting levels in the entire group and absolute changes above baseline in the athlete group showed that: (1) no significant relationships exist between serum and salivary IL-6 levels; (2) the greater the salivary IL-6 measurement, the higher the resultant inaccuracy of the SA method; (3) significant correlations exist between isometric force and mechanical fatigue during exercise and peaks of lactate and serum IL-6. These data provided demonstration of a cotton-interference effect for the results of salivary IL-6 assay and confirmed the lack of significant correlation between salivary and serum IL-6 in resting and post-exercise conditions.     

Transplantation in the onco-hematology field: focus on the manipulation of αβ and γδ T cells

γ/δ T cells represent a subset of T cells expressing a T cell receptor (TCR) variant composed of gamma and delta chains. The γ/δ TCR is expressed by 2-10% of all T cells in human peripheral blood, whereas the majority of T cells express α/β TCRs. γ/δ T cells display a range of innate effector functions including rapid secretion of chemokines and cytokines, as well as target cell lysis. Recent interest has focused on the function of γ/δ T lymphocytes in allogeneic transplantation in the onco-hematology field. Several studies, in vitro and in vivo, suggest that γ/δ T lymphocytes are potential beneficial effector cells in the context of hematopoietic stem cell transplantation (HSCT). In addition, in this review, we discuss the depletion of α/β T lymphocytes in the graft for allogeneic transplantation. In fact, an efficient TCR α/β cell depletion potentially reduces the risk of GvHD. Furthermore, TCR α/β T cell depletion, especially with immunomagnetic negative selection, retains other potential beneficial effector cells in the graft, such as γ/δ T cells, NK cells, and stem cells. These "facilitating" cells might facilitate engraftment, exert GvL effects, and reduce the risk for infections.     

The genetic and molecular basis of muscular dystrophy: roles of cell–matrix linkage in the pathogenesis

Muscular dystrophies are a heterogeneous group of genetic disorders. In addition to genetic information, a combination of various approaches such as the use of genetic animal models, muscle cell biology, and biochemistry has contributed to improving the understanding of the molecular basis of muscular dystrophy's etiology. Several lines of evidence confirm that the structural linkage between the muscle extracellular matrix and the cytoskeleton is crucial to prevent the progression of muscular dystrophy. The dystrophin–glycoprotein complex links the extracellular matrix to the cytoskeleton, and mutations in the component of this complex cause Duchenne-type or limb-girdle-type muscular dystrophy. Mutations in laminin or collagen VI, muscle matrix proteins, are known to cause a congenital type of muscular dystrophy. Moreover, it is not only the primary genetic defects in the structural or matrix proteins, but also the primary mutations of enzymes involved in the protein glycosylation pathway that are now recognized to disrupt the matrix–cell interaction in a certain group of muscular dystrophies. This group of diseases is caused by the secondary functional defects of dystroglycan, a transmembrane matrix receptor. This review considers recent advances in understanding the molecular pathogenesis of muscular dystrophies that can be caused by the disruption of the cell–matrix linkage.     

A genome-wide analysis of the molecular alterations occurring in the adenomatous and carcinomatous components of the same tumor based on the adenoma–carcinoma sequence

Identification of molecular alterations occurring in the adenomatous and carcinomatous components within the same tumor would greatly enhance understanding of the neoplastic progression of colorectal cancer. We examined somatic copy number alterations (SCNAs) and mRNA expression at the corresponding loci involved in the adenoma–carcinoma sequence in the isolated adenomatous and cancer glands of the same tumor in 15 cases of microsatellite-stable “carcinoma in adenoma,” using genome-wide SNP and global gene expression arrays. Multiple copy-neutral loss of heterozygosity events were detected at 4q13.2, 15q15.1, and 14q24.3 in the adenomatous component and at 4q13.2, 15q15.1, and 14q24.3 in the carcinomatous component. There were significant differences in the copy number (CN) gain frequencies at 20q11.21–q13.33, 8q13.3, 8p23.1, and 8q21.2–q22.2 between the adenomatous and carcinomatous components. Finally, we found a high frequency of five genotypes involving CN gain with upregulated expression of the corresponding gene (RPS21, MIR3654, RSP20, SNORD54, or ASPH) in the carcinomatous component, whereas none of these genotypes were detected in the adenomatous component. This finding is interesting in that CN gain with upregulated gene expression may enhance gene function and play a crucial role in the progression of an adenoma into a carcinomatous lesion.     

Nail–Patella Syndrome: clinical and molecular data in 55 families raising the hypothesis of a genetic heterogeneity

Nail–Patella Syndrome (NPS) is a rare autosomal dominant condition comprising nail and skeletal anomalies. Skeletal features include dysplastic patellae and iliac horns, as well as scapula and elbow dysplasia. Nephropathy and glaucoma or intra-ocular hypertension can sometimes be present. NPS is due to variants affecting function in LMX1B, which encodes a LIM-homeodomain protein critical for limb, kidney and eye development. We describe the phenotype and the molecular data of 55 index patients and their 39 relatives presenting with typical NPS. We identified 38 different LMX1B anomalies, 19 of which were not reported before. In our series, 9% of families are not carriers of a LMX1B genomic alteration after extensive study of the coding and non-coding regions of the gene. One of the families showed no linkage to the LMX1B locus, raising the hypothesis of a genetic heterogeneity.     

Short- and long-term effects of tactile massage on salivary cortisol concentrations in Parkinson’s disease: a randomised controlled pilot study

Background

Parkinson’s disease (PD) is a chronic neurodegenerative disorder with limited knowledge about the normal function and effects of non-pharmacological therapies on the hypothalamic-pituitary-adrenal (HPA) axis. The aim of the study was to analyse the basal diurnal and total secretion of salivary cortisol in short- and long-term aspects of tactile massage (TM)_.

Methods

Design: Prospective, Controlled and Randomised Multicentre Trial.Setting and interventions: Forty-five women and men, aged 50–79 years, were recruited. Twenty-nine of them were blindly randomised to tactile massage (TM) and 16 of them to the control group, rest to music (RTM). Ten interventions were given during 8 weeks followed by a 26 weeks of follow up. Salivary cortisol was collected at 8 am, 1 pm, 8 pm, and 8 am the next day, on five occasions. With the first and eighth interventions, it was collected immediately before and after intervention.Main outcome measures: The primary aim was to assess and compare cortisol concentrations before and immediately after intervention and also during the follow-up period. The secondary aim was to assess the impact of age, gender, body mass index (BMI), duration and severity of PD, effects of interventional time-point of the day, and levodopa doses on cortisol concentration.

Results

The median cortisol concentrations for all participants were 16.0, 5.8, 2.8, and 14.0 nmol/L at baseline, later reproduced four times without significant differences. Cortisol concentrations decreased significantly after TM intervention but no change in diurnal salivary cortisol pattern was found. The findings of reduced salivary cortisol concentrations immediately after the interventions are in agreement with previous studies. However, there was no significant difference between the TM and control groups. There were no significant correlations between cortisol concentrations and age, gender, BMI, time-point for intervention, time interval between anti-parkinson pharmacy intake and sampling, levodopa doses, duration, or severity of PD.

Conclusions

Diurnal salivary cortisol rhythm was normal. Salivary cortisol concentrations were significantly reduced after the TM intervention and after RTM, but there were no significant differences between the groups and no sustained long-term effect. No associations were seen between salivary cortisol concentration and clinical and/or pharmacological characteristics.

Trial registration

ClinicalTrial.gov, NCT01734876 and FoU Sweden 108881.

     

Associations between patient–provider communication and socio-cultural factors in prostate cancer patients: A cross-sectional evaluation of racial differences

Objective

To examine the association between socio-cultural factors and patient–provider communication and related racial differences.

Methods

Data analysis included 1854 men with prostate cancer from a population-based study. Participants completed an assessment of communication variables, physician trust, perceived racism, religious beliefs, traditional health beliefs, and health literacy. A multi-group structural equation modeling approach was used to address the research aims.

Results

Compared with African Americans, Caucasian Americans had significantly greater mean scores of interpersonal treatment (p < 0.01), prostate cancer communication (p < 0.001), and physician trust (p < 0.001), but lower mean scores of religious beliefs, traditional health beliefs, and perceived racism (all p values <0.001). For both African and Caucasian Americans, better patient–provider communication was associated with more physician trust, less perceived racism, greater religious beliefs (all p-values <0.01), and at least high school education (p < 0.05).

Conclusion

Socio-cultural factors are associated with patient–provider communication among men with cancer. No evidence supported associations differed by race.

Practice implication

To facilitate patient–provider communication during prostate cancer care, providers need to be aware of patient education levels, engage in behaviors that enhance trust, treat patients equally, respect religious beliefs, and reduce the difficulty level of the information.     

Angiogenesis of glioma: evaluation of ultrastructural characteristics of microvessels and tubular bodies (Weibel–Palade) in endothelial cells and immunohistochemical findings with VEGF and p53 protein

Capillary endothelial proliferation is often a prominent feature of malignant gliomas. The understanding of structural and functional characteristics of the vascular microenvironment in gliomas is essential for the design of future therapeutic strategies against this tumor. Electron microscopic analysis of the capillary endothelial proliferation in malignant gliomas indicated that the complex vascular structures within the tumor were composed essentially of immature capillaries. Immature capillaries had a narrow slitlike lumen composed of endothelial cells with their high nuclear:cytoplasmic ratio and the relative paucity of organelles. They resembled capillary buds seen in normal repair tissue. Immature microvessels caused by angiogenesis were found more frequently in marginal zone of the tumors with increased microvessels. The tubular body was an organelle observed in vascular endothelial cells and was used frequently as a marker of the endothelial cell. Tubular bodies were evaluated by quantitative measurement of the mean percent (%) ratio of the number of endothelial cells with tubular bodies to all endothelial cells in microvessels of tumors. In glioblastomas it yielded a value of 32.4% in the margin, about two times as high as that in the center of the tumors. However, it was lower in all locations of astrocytomas. Tubular bodies in endothelial cells could be increased in proportion to neovascularization, and they might serve as a marker for increasing microvessels in astrocytic tumors. Tumor angiogenesis may be regulated by growth factors with angiogenic activities that are secreted by tumor cells. Vascular endothelial growth factor (VEGF) may play a critical role in the regulation of vascular endothelial proliferation. We found that 86% of 29 glioblastomas and 79% of 14 anaplastic astrocytomas demonstrated immunoreactivity for VEGF in their tumor cells. There tended to be a correlation between VEGF and vascularity. A correlation existed between the grade of immunoreactivity for VEGF and the grade of p53 protein expression in the malignant gliomas. However, the MIB-1 indices did not increase in correlation with increase in the extent of immunoreactivity for VEGF.     

Expression of aquaporin-5 in and fluid secretion from immortalized human salivary gland ductal cells by treatment with 5-aza-2'-deoxycytidine: a possibility for improvement of xerostomia in patients with Sjögren's syndrome

The aim of the present study was to investigate the possibility that ductal cells, which preferentially survive and/or proliferate in Sj?gren's syndrome (SS) salivary glands of patients with SS, could acquire the functional expression of membrane water channel aquaporin-5 (AQP5). Thus, in this study, we demonstrate that an immortalized normal human salivary gland ductal cell (NS-SV-DC) line, lacking the expression of AQP5, acquires AQP5 gene expression in response to treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA demethylating agent. Confocal microscopic analysis revealed the localization of AQP5 expression mainly at the apical and lateral sides of the plasma membrane. The expressed AQP5 protein was functionally active because AQP5 expression resulted in a significant increase in the osmotically directed net fluid rate across monolayers of NS-SV-DC cells. By the analysis of bisulfite sequencing of CpG islands in the AQP5 promoter, hypermethylation within the consensus Sp1-binding sites was commonly observed in parental cell clones, whereas demethylation at the CGs, one in the second consensus Sp1 element and the other outside of the third consensus Sp1 element in the AQP5 promoter, was detected in NS-SV-DC cells after treatment with 5-Aza-CdR. By analyzing the luciferase activity of transfected AQP5 promoter vectors, it became evident that demethylation at the CGs cooperatively functions between these two sites to induce AQP5 expression. Our data, therefore, suggest that treatment of ductal cells with 5-Aza-CdR could result in the expression of the AQP5 gene, thereby leading to increased fluid secretion from ductal cells in SS salivary glands.     

The grey zone between pure (neuro)endocrine and non-(neuro)endocrine tumours: a comment on concepts and classification of mixed exocrine–endocrine neoplasms

Terms such as “mixed endocrine–exocrine carcinoma” (MEEC) and “adenocarcinoma with neuroendocrine (NE) differentiation” (ADC-NE) identify tumours belonging to the spectrum of neoplasms with divergent exocrine and (neuro)endocrine differentiation. These tumours display variable quantitative extent of the two components, potentially ranging from 1 to 99%, and variable structural patterns, ranging from single scattered NE cells to a well-defined NE tumour cell population organized in organoid, trabecular or solid growth patterns. In the present report, the grey zone of tumours/carcinomas with mixed NE and non-NE features is explored for various organs. From a practical point of view, MEECs differ from carcinomas with focal NE differentiation by (1) the extension of each component (more than 30%) and (2) the structural pattern of the NE component, either organoid for well-differentiated or solid/diffuse for poorly differentiated cases. In MEECs, the most aggressive cell population drives the clinical behaviour. Conversely, ADC-NE generally do not show a different clinical outcome, compared to the corresponding conventional forms, except for prostatic adenocarcinoma, in which NE cells are a negative prognostic factor. The recognition of MEECs may be of relevance for a targeted therapeutic strategy, foreseeing the use of biotherapies similar to those proposed for pure NE tumours.