Ligand-dependent Hedgehog pathway activation in Rhabdomyosarcoma: the oncogenic role of the ligands.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. The Hedgehog (HH) pathway is known to develop an oncogenic role in RMS. However, the molecular mechanism that drives activation of the pathway in RMS is not well understood. METHODS: The expression of HH ligands was studied by qPCR, western blot and immunohistochemistry. Functional and animal model studies were carried out with cells transduced with shRNAs against HH ligands or treated with HH-specific inhibitors (Vismodegib and MEDI-5304). Finally, the molecular characterisation of an off-target effect of Vismodegib was also made. RESULTS: The results showed a prominent expression of HH ligands supporting an autocrine ligand-dependent activation of the pathway. A comparison of pharmacologic Smoothened inhibition (Vismodegib) and HH ligand blocking (MEDI-5304) is also provided. Interestingly, a first description of pernicious off-target effect of Vismodegib is also reported. CONCLUSIONS: The clarification of the HH pathway activation mechanism in RMS opens a door for targeted therapies against HH ligands as a possible alternative in the future development of better treatment protocols. Moreover, the description of a pernicious off-target effect of Vismodegib, via unfolded protein response activation, may mechanistically explain its previously reported inefficiency in several ligand-dependent cancers.

MYCN repression of Lifeguard/FAIM2 enhances neuroblastoma aggressiveness.

Neuroblastoma (NBL) is the most common solid tumor in infants and accounts for 15% of all pediatric cancer deaths. Several risk factors predict NBL outcome: age at the time of diagnosis, stage, chromosome alterations and MYCN (V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma-Derived Homolog) amplification, which characterizes the subset of the most aggressive NBLs with an overall survival below 30%. MYCN-amplified tumors develop exceptional chemoresistance and metastatic capacity. These properties have been linked to defects in the apoptotic machinery, either by silencing components of the extrinsic apoptotic pathway (e.g. caspase-8) or by overexpression of antiapoptotic regulators (e.g. Bcl-2, Mcl-1 or FLIP). Very little is known on the implication of death receptors and their antagonists in NBL. In this work, the expression levels of several death receptor antagonists were analyzed in multiple human NBL data sets. We report that Lifeguard (LFG/FAIM2 (Fas apoptosis inhibitory molecule 2)/NMP35) is downregulated in the most aggressive and undifferentiated tumors. Intringuingly, although LFG has been initially characterized as an antiapoptotic protein, we have found a new association with NBL differentiation. Moreover, LFG repression resulted in reduced cell adhesion, increased sphere growth and enhanced migration, thus conferring a higher metastatic capacity to NBL cells. Furthermore, LFG expression was found to be directly repressed by MYCN at the transcriptional level. Our data, which support a new functional role for a hitherto undiscovered MYCN target, provide a new link between MYCN overexpression and increased NBL metastatic properties.

Targeting the voltage-dependent K(+) channels Kv1.3 and Kv1.5 as tumor biomarkers for cancer detection and prevention.

Potassium channels (KCh) are a diverse group of membrane proteins that participate in the control of the membrane potential. More than eighty different KCh genes have been identified, which are expressed in virtually all living cells. In addition to nerve and cardiac action potentials, these proteins are involved in a number of physiological processes, including cell volume regulation, apoptosis, immunomodulation and differentiation. Furthermore, many KCh have been reported to play a role in proliferation and cell cycle progression in mammalian cells, and an important number of studies report the involvement of KCh in cancer progression. The voltage dependent potassium (Kv) channels, in turn, form the largest family of human KCh, which comprises about 40 genes. Because Kv1.3 and Kv1.5 channels modulate proliferation of different mammalian cells, these proteins have been analyzed in a number of tumors and cancer cells. In most cancers, the expression patterns of Kv1.3 and Kv1.5 are remodeled, and in some cases, a correlation has been established between protein abundance and grade of tumor malignancy. The list of cancers evaluated is constantly growing, indicating that these proteins may be future targets for treatment. The aim of this review is to provide an updated overview of Kv1.3 and Kv1.5 channels during cancer development. Unlike Kv1.5, Kv1.3 is characterized by a very selective and potent pharmacology, which could lead to specific pharmacological targeting. Because potassium channels may play a pivotal role in tumor cell proliferation, these proteins should be taken into account when designing new cancer treatment strategies.

Voltage-dependent potassium channels Kv1.3 and Kv1.5 in human cancer.

Membrane ion channels participate in cancerous processes such as proliferation, migration and invasion, which contribute to metastasis. Increasing evidence indicates that voltage-dependent K(+) (Kv) channels are involved in the proliferation of many types of cells, including tumor cells. Kv channels have generated immense interest as a promising tool for developing new anti-tumor therapies. Therefore, the identification of potential biomarkers and therapeutic targets in specific cancers is an important prerequisite for the treatment. Since Kv1.3 and Kv1.5 are involved in the proliferation of many mammalian cells, we aimed to study the expression of Kv1.3 and Kv1.5 in a plethora of human cancers. Thus, tissues from breast, stomach, kidney, bladder, lung, skin, colon, ovary, pancreas, brain, lymph node, skeletal muscle and some of their malignant counterparts have been analyzed. Whereas Kv1.3 expression was either decreased or did not change in most tumors, Kv1.5 was overexpressed. However, the presence of Kv1.3 was mostly associated with inflammatory lymphoplasmocytic cells. Independent of the suitability of individual channels as therapeutic targets, the identification of a Kv phenotype from tumor specimens could have a diagnostic value of its own. Our results demonstrate that Kv1.5, and to some extent Kv1.3, are aberrantly expressed in a number of human cancers. These channels could serve both as novel markers of the metastatic phenotype and as potential new therapeutic targets. The concept of Kv channels as therapeutic targets or prognostic biomarkers attracts increasing interest and warrants further investigation.

Fetal and placenta chromosome constitution in 237 pregnancy losses.

The aim of the study was to carry out cytogenetic analyses in pregnancy losses. Samples of cartilage and placenta tissue were obtained prospectively from 237 pregnancy losses of more than 16 weeks of gestation (130 stillbirths, 97 induced abortions and 10 early neonatal deaths). Cartilage culture was performed in 222 samples and placental culture was initiated in 224. The overall culture success rate was 83.5%, 72.3% in stillbirths, 97% in induced abortions and 100% in early neonatal death. An abnormal karyotype was detected in 52 cases: 6.9% in stillbirths, 43.6% in induced abortions and 20% in early neonatal deaths. The rate of discrepancy between the prenatal cytogenetic results in amniotic fluid and the post-termination karyotype was 3%. The tissue of choice for cytogenetic analysis was cartilage in induced abortions and early neonatal death, and placenta in stillbirth. The majority of cases had a chromosome abnormality: multiple congenital anomalies in 74.6%, and a single major anomaly in 9.7%.

Increased protein levels of heterogeneous nuclear ribonucleoprotein A2/B1 in fetal Down syndrome brains.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are predominantly nuclear RNA-binding proteins that form complexes with RNA polymerase II transcripts. These proteins play pivotal roles in transcription, pre-mRNA processing in the nucleus, cytoplasmic mRNA translation and its turnover. In addition, hnRNPs have been shown to be essential for embryonic development of Drosophila. Here we studied the protein levels of hnRNPs (A2/B1, H and H') in fetal brain with Down syndrome (DS; n = 5) compared to controls (n = 7). We used two-dimensional (2-D) gel electrophoresis, matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) and specific software for quantification. hnRNP A2/B1 was significantly increased in fetal DS brain (13.52+/-4.50) compared to controls (9.16+/-1.35), but both hnRNP H and H' were unchanged. Increased hnRNP A2/B1 in fetal DS brain may represent high activity of RNA processing such as RNA trafficking and telomere protection, and/or it could contribute to abnormal development of DS brains. Furthermore, comparable expression of hnRNP H and H' suggest a specific upregulation of hnRNP A2/B.

Expression profiles of proteins in fetal brain with Down syndrome.

Proteomics is a powerful tool for evaluating differential protein expression comparing hundreds of proteins simultaneously. In the current study we performed "gene hunting" at the protein level and identified and quantified 10 protein spots in control and Down syndrome (DS) fetal brains. Using two-dimensional (2-D) electrophoresis of fetal brain proteins with subsequent MALDI-identification and quantification with specific software, we identified a series of poorly known proteins, in part hypothetical and orphans or poorly documented proteins. Hypothetical protein DKFZp564D177.1-human (fragment), one of these proteins was identified in fetal brain and was significantly decreased in DS (0.61+/-0.44, n = 7) compared to controls (3.43+/-1.83, n = 7). Septin 6, previously shown to be associated with synaptic vesicles, was present in all of 7 controls, but only in 1 out of 6 DS brains. We suggest that decreased protein levels of hypothetical protein DKFZp564D177.1-human (fragment) and lower prevalence of septin 6 could be involved in the maldevelopment of fetal DS brains. The other 8 proteins (WD repeat protein 1, novel protein highly similar to septin 2 homolog, septin 5, septin 2, DJ37E16.5 (novel protein similar to nitrophenylphosphatases from various organism), hypothetical 30.2 kDa protein, neuronal protein NP25, and DC7 protein (vacuolar sorting protein 29)) were comparable between controls and DS but could be identified in fetal and DS cortex, thus proposing them as tentative brain proteins.

Cellulose granulomatosis of the lungs.

Chest radiographs and high-resolution chest CT scans were performed in a 30-year-old man with a history of intravenous drug abuse and diffuse micronodular infiltrates. Transbronchial biopsy gave a diagnosis of cellulose granulomatosis of the lung. Cellulose granulomatosis should be considered in the differential diagnosis of pulmonary interstitial disease, especially in the setting of intravenous drug abuse.

Prognostic analysis of astrocytic gliomas correlating histological parameters with the proliferating cell nuclear antigen labelling index (PCNA-LI).

Eighty out of 250 cases of astrocytic glioma collected from a practice served by a single clinical team over a 15-year period were studied using a full complement of clinical, follow up, histopathological analysis and proliferating cell nuclear antigen (PCNA) immunostaining for the obtention of the PCNA-labelling index (LI). A statistical evaluation and discriminant analysis were carried out with the aim of clarifying the importance of various parameters as predictors of tumor behaviour. Data are correlated with survival (with a 10-year follow up). A significant correlation with survival was found when histological grouping and the PCNA-LI were studied with the Cox test. Most significant features were histological as detected using classical techniques including histological grading. The utilization of objective values (mitosis, cellular density and necrosis) appears to be useful in grading astrocytic tumors. Our results emphasize the importance of cytological, histological and PCNA-LI parameters as predictors of tumor behaviour.

Argyrophilic nucleolar organizer region (AgNOR) counting in astrocytic gliomas: prognostic value.

In 87 astrocytic gliomas the number of AgNORs/nucleus was retrospectively studied and data correlated with the histological type of the tumors and survival. All patients were treated by the same surgical team and with uniform criteria. Statistically significant differences (p < 0.01) were found in relation with the AgNOR averages among the histological types of tumors. A statistically significant linear correlation (p < 0.05) between the AgNOR values and survival of the patients was also found. Patients with mean AgNOR values higher than 2.23 and lower than 2.9 survived an average of 11.5 +/- 9.1 months vs. a survival in average of 24.4 +/- 34.1 months with mean AgNOR values under 2.23 (p < 0.05). Patients with AgNOR values higher than 2.9 survived, on average, 7.7 +/- 3.9 months. AgNOR counting in astrocytic gliomas is a reproducible, easy, quick method with prognostic value. AgNORs may be successfully applied in routine material to assess the growth potential of astrocytic gliomas.

Invasive aspergillosis: a life-threatening complication of short-term steroid treatment.

OBJECTIVE: To describe a patient with invasive pulmonary aspergillosis related to short-term steroid treatment. CASE SUMMARY: A 78-year-old man with chronic obstructive pulmonary disease (COPD) developed an invasive pulmonary aspergillosis after short-term (less than 1 week) intravenous steroid therapy. The diagnosis was established by recovering Aspergillus fumigatus from a bronchoalveolar lavage and was confirmed by autopsy, with the additional finding of an aspergilloma. DISCUSSION: This case is of interest for 3 reasons: (1) it illustrates that invasive aspergillosis may be followed by a rapidly progressive respiratory failure, even in the absence of a fever; (2) this patient had simultaneously an aspergilloma and an invasive aspergillosis; and (3) it confirms reports indicating that short-term steroid therapy for COPD represents a significant risk factor for opportunistic lung infections. CONCLUSIONS: In patients with COPD who receive even short-term steroid therapy and who have progressive respiratory failure caused by pneumonia, invasive aspergillosis should be suspected early and acted upon accordingly.

Primary leptomeningeal lymphoma presenting as cerebellopontine angle lesion.

We report a primary leptomeningeal lymphoma (PLML) presenting as a cerebellopontine angle lesion. CT showed slight enlargement of the ventricular system, obliteration of the basal cisterns and a dense lesion in the left cerebellopontine angle which enhanced with contrast medium. Cerebrospinal fluid abnormalities included sterile lymphocytic pleocytosis without malignant cells, low sugar and high adenosine deaminase levels. An erroneous diagnosis of tuberculous meningitis was made, but autopsy revealed a leptomeningeal B-cell lymphoma with infiltration of the middle cerebellar peduncle giving the appearance of a cerebellopontine angle lesion. Seven cases of cerebellopontine angle lymphoma have previously been described, only one of which could be classified as PLML.

Aortic bypass graft infection due to Aspergillus: report of a case and review.

Aspergillus is a rare cause of aortic graft infection. A recent case is reported and a review of seven other cases described in the literature since 1966 is presented. Infections of both thoracic and abdominal aortic grafts have been reported. Infection occurred from 5 weeks to 3 years after surgery. Underlying immunosuppressive disorders were not present. The most common presenting symptoms were back pain, fever, and embolic phenomena. Pseudoaneurysm of the vascular prosthesis with contiguous vertebral osteomyelitis was frequently seen. Blood cultures were always negative. Laboratory findings were nonspecific. The diagnosis was not anticipated in any case. Aspergillus was isolated in culture of specimens of the vertebral bone, excised graft, or peripheral emboli. Aspergillus fumigatus was the species most frequently isolated. Infection may have occurred intraoperatively as a result of contamination with airborne fungal spores. Optimal treatment included early removal of the graft with extraanatomical bypass plus prolonged antifungal therapy. Delayed surgical intervention and medical therapy alone were associated with high mortality rates. Aspergillar vascular infection should be suspected in patients with aortic grafts who develop persistent back pain, fever, or arterial embolization, and whose blood cultures are sterile.

Epithelial differentiation in gliomas, meningiomas and choroid plexus papillomas.

The immunohistological findings using antibodies to different intermediate filaments (glial fibrillary acidic protein, vimentin and two types of cytokeratin) and epithelial membrane antigen are described in 89 gliomas, 19 meningiomas and 8 choroid plexus papillomas (CPPs) from adult patients. All the patients had total or subtotal surgical excision of their tumours with clinical follow up for between 3 and 7 years. The immunohistological results were correlated with the histological features and patient survival. Tumours other than low grade astrocytomas, oligodendrogliomas and anaplastic ependymomas expressed one or more epithelial markers. This immunohistological evidence of epithelial differentiation in the absence of histological epithelial features in gliomas confirms that the two are not necessarily correlated. It is concluded that the expression of epithelial markers in some intradural tumours may reflect aberrant differentiation related to the degree of anaplasia in poorly differentiated astrocytomas and glioblastomas. All the patients with anaplastic epithelial marker-positive gliomas died within 1 year, whereas only 68% of patients with marker-negative tumours died within the follow-up period. In ependymomas and meningiomas, the expression of epithelial markers may reflect their histogenesis, while in malignant CPPs such expression could denote either their aberrant differentiation or histogenetic derivation.

Helicobacter pylori in Barrett's esophagus.

Barrett's esophagus is an anatomicoclinical state in which, due to the prolonged action of gastroesophageal reflux, the squamous epithelium is replaced by columnar epithelium. Helicobacter pylori has been implicated in the pathogenesis of various gastrointestinal disorders and has occasionally been observed in Barrett's esophagus. The aim of this study is to determine the incidence of H. pylori in Barrett's esophagus and try to establish its role in the pathogenesis of this disorder. H. pylori was observed in 31 biopsies (44.3%) of the 70 studied, mainly when the epithelium is of the gastric atrophic-fundic type (p less than 0.01). Its presence shows no relation to the degree of inflammatory activity and does not seem, therefore, to play an important role in the pathogenesis of the lesion.