Role of neurotrophins and their receptors in human neuroblastomas: a primary culture study

Expression of trk family genes are prognostic indicators of neuroblastoma. However, the functional role of neurotrophins and their receptors in neuroblastomas in vivo is still unclear. We studied the expression of neurotrophin receptors (trk-A, trk-B, trk-C) and their responsiveness to neurotrophins (NGF, BDNF, NT-3) in 25 human neuroblastomas using a primary culture system. The tumours in early stages and stage 4s responded to both NGF and NT-3, but not to BDNF, by surviving and differentiating terminally and the responsiveness was correlated with high levels of trk-A, especially the neuronal isoform. However, in many advanced stage tumours, the expression of trk-A was down-regulated and the response pattern to neurotrophins was diverse, without showing terminal differentiation. Interestingly, a stage 4 tumour with MYCN amplification which expressed high level of neuronal trk-A was dependent on nerve growth factor (NGF) for both survival and differentiation in primary culture. The results suggest that the NGF/trk-A signalling may be the main regulatory pathway for differentiation and survival of neuroblastoma in vivo and that trk-A overexpression may overcome aggressiveness, even of the tumour with MYCN amplification.     

Somatostatin in neuroblastoma and ganglioneuroma

Neuroblastoma, a childhood tumour of the sympathetic nervous system, may in some cases differentiate to a benign ganglioneuroma or regress due to apoptosis. Somatostatin may inhibit neuroblastoma growth and induce apoptosis in vitro and was therefore investigated. Using a radioimmunoassay, we found that all ganglioneuromas contained high somatostatin concentrations (> 16 pmol/g), significantly higher than neuroblastomas (n = 117, median 2.8 pmol/g), healthy adrenals, Wilms' tumours, phaeochromocytomas and other neuroendocrine tumours (P < 0.001). Neuroblastomas contained more somatostatin than control tumours (P < 0.001-0.05). Neuroblastomas amplified for the MYCN oncogene contained less somatostatin than non-amplified tumours (1.2 pmol/g versus 4.0 pmol/g, respectively; P = 0.026). In a clinically unfavourable neuroblastoma subset (age > 12 months, stage 3 or 4) 16 children with high concentrations of somatostatin in primary tumours had a better prognosis than 23 with low somatostatin (46.7% versus 0% survival at 5 years, P < 0.005). Scintigraphy using 111In-pentetreotide identified tumours expressing high-affinity somatostatin receptors in vivo. However, no significant correlation was found between somatostatin receptor expression and peptide content in 15 tumours. Similarly, human SH-SY5Y neuroblastoma xenografts grown in nude rats showed low somatostatin concentrations, but were positive for somatostatin receptor scintigraphy. Treatment of these rats with the somatostatin analogue octreotide seemed to upregulate in vivo receptor expression of somatostatin and vasoactive intestinal peptide more effectively than 13-cis retinoic acid. In conclusion, somatostatin in neuroblastoma is associated with differentiation to benign ganglioneuromas in vivo and favourable outcome in advanced tumours. Furthermore, somatostatin receptor scintigraphy may identify tumours with high-affinity receptors in children that might benefit from targeted therapy using synthetic somatostatin analogues.     

Telomerase activity in neuroblastoma: is it a prognostic indicator of clinical behaviour?

Neuroblastomas show remarkable biological heterogeneity, resulting in favourable prognosis or unfavourable prognosis due to aggressive growth despite multimodal therapy. Recently, we proposed that aggressive tumours express telomerase at a high level while the favourable tumours lack or have low telomerase expression. To evaluate the correlation between telomerase activity and other biological characteristics reported as prognostic markers (MYCN gene amplification, loss of heterogeneity (LOH) in the short arm of chromosome 1, trk-A expression, Ha-ras p21 expression, and DNA ploidy), we investigated these biological features in 105 untreated neuroblastomas. In these cases, 23 showed high telomerase activity, 78 showed low activity, and telomerase activity was undetectable in 4 cases. Most tumours with genetic alterations (MYCN amplification or 1p32 LOH) showed high telomerase activity. Most tumours with low or undetectable activity were aneuploid, and showed trk-A and Ha-ras expression. Three of the four tumours with undetectable telomerase activity regressed. In 2 of the tumours with low telomerase activity, the residual tumours maturated and showed repression of telomerase activity. Thus, the level of telomerase activity correlated with other genetic alterations and/or gene expression and may be a useful prognostic indicator in neuroblastoma.     

Monoclonal antibody to human Trk-A: diagnostic and therapeutic potential in neuroblastoma

5C3 is a murine IgG1 antibody specific for the nerve growth factor (NGF) docking site of the human p140 trk-A receptor, with no cross-reactivity with human trk-B. In vitro, 5C3 and its Fab mimic the effects of NGF, a neurotrophin mediating growth and differentiation of neural crest-derived cells. When labelled with radioisotope, 5C3 images human trk-A positive tumours in vivo. More importantly, 5C3 induces regression of human trk-A positive tumours in rodents. We therefore investigated the value of 5C3 in detecting trk-A expression in human neuroblastoma by immunohistochemistry. 5C3 reactivity was detected in 73 of 113 neuroblastoma specimens and correlated strongly with localised/4s disease (55/60) with either a homogeneous or mixed pattern. Among stage 4 neuroblastoma, only 18/53 had homogeneous or mixed trk-A expression. 5C3 did not react with 46/48 other human malignancies, but was positive in 1 melanoma and 1 Wilms' tumour specimen. The prognostic, imaging and NGF-mimetic properties of antibody 5C3 and its derivatives may offer alternatives for the diagnosis and treatment of neuroblastoma.     

Activation of trk-A but not trk-B signal transduction pathway inhibits growth of neuroblastoma cells

In neuroblastoma tumours, the expression of high levels of trk-A mRNA, which encodes the high-affinity nerve growth factor (NGF) receptor, is associated with good prognosis. Constitutive expression of brain-derived neurotrophic factor (BDNF) and variable expression of its receptor trk-B are frequently detected in tumours from patients with a poor prognosis. To evaluate the biological consequences of activation of the trk-A or trk-B signal transduction pathways in neuroblastoma cells, the trk-A or trk-B gene was transfected into the trk negative 15N neuroblastoma cell line. Clones expressing trk-A or trk-B were treated with specific ligands and evaluated for growth and differentiation. Both ligands induced neurite extension. Treatment of the 15N-trk-A clones with NGF inhibited proliferation (80-90% decrease), while treatment of the 15N-trk-B clone with BDNF had no effect (< 10% decrease). NGF-induced growth inhibition was concentration dependent. Such studies indicate that differential trk expression may affect the biology of neuroblastoma tumours and contribute to differences in the clinical course of patients.     

Interleukin-1 beta converting enzyme (ICE) is preferentially expressed in neuroblastomas with favourable prognosis

To determine whether interleukin-1 beta converting enzyme (ICE) plays a role in the programmed cell death of neuroblastoma, we studied ICE expression in primary tumours. In patients in stages I, II and IVS, ICE mRNA was detected in 22 of 32 (69%) tumours, while only 5 of 26 (19%) tumours expressed ICE in stages III and IV (P < 0.001). ICE mRNA was expressed in 27 of 47 (57%) tumours without MYCN amplification, but it was not detected in any tumours with MYCN amplification (P < 0.01). Immunohistochemically, the cytoplasm was stained in all 15 neuroblastomas examined. The nuclei were stained in 12 neuroblastomas without MYCN amplification, whereas only 1 of 3 tumours with MYCN amplification had positive staining in the nuclei. In ganglioneuromas, high levels of ICE mRNA were expressed, but immunostaining showed that the protease expression was confined to the cytoplasm. These observations suggest that ICE may be associated with the spontaneous regression often seen in favourable neuroblastomas and that localisation of ICE protease in the cell may be important for the cell death pathway. Double staining for ICE and TUNEL showed that they were co-localised in some nuclei, but the distribution of ICE protease expression was not necessarily the same as that of DNA fragmentation, suggesting that the protease expression probably preceded DNA fragmentation during the apoptotic process. ICE may play an important role in regulating the apoptotic process of neuroblastoma.     

Diagnostic value of tissue polypeptide-specific antigen (TPS) in neuroblastoma and Wilms' tumour

Although tissue polypeptide-specific antigen (TPS) has been described as a potentially useful serum marker of tumour activity in adult epithelial tumours, few data are available for childhood malignancies. Neuroblastomas and Wilms' tumours are the commonest types of solid malignancies found in the retroperitoneum of children. At this time, a widely used marker for Wilms' tumour is not available. Using an enzyme-linked immunosorbent assay (ELISA) kit, serum TPS levels in 23 children with neuroblastomas, nine with Wilms' tumours and 22 with benign tumours were evaluated to test the usefulness of the marker in identifying malignancies. Compared with healthy children (n = 110), the preoperative least-square means (LSM) of serum TPS were considerably elevated in both neuroblastoma (LSM = 209 U l(-1)) and Wilms' tumour (LSM = 235 U l(-1)), whereas values in benign tumours were only slightly elevated. Although the Wilms' tumours were associated with higher preoperative serum TPS levels, there was no statistically significant difference compared with neuroblastomas. Receiver operating characteristic analysis (ROC curves) showed a high sensitivity and specificity for both malignancies. Successful treatment resulted in decrease in TPS serum values. Serum TPS measurements in children presenting with abdominal masses can help in diagnosing the two commonest extracranial solid malignancies of childhood. Furthermore, TPS could acquire a pivotal role in monitoring therapy.     

Comparison of a videokeratoscope and an autokeratometer as predictors of the optimum back surface curves of rigid corneal contact lenses

Vasoactive intestinal polypeptide (VIP) is reported to exert an autocrine control on neuroblastoma cell tumours: VIP is produced by the tumour and stimulates cell differentiation. This study tested the hypothesis that the parent peptide; the pituitary adenylate cyclase activating polypeptide (PACAP) may have a similar role. It was found that PACAP mRNA and PACAP were expressed in 12/12 tumours; it was also observed that PACAP receptor mRNA and functional PACAP receptors were expressed in 12/12 and 5/9 tumours, respectively. VIP mRNA and VIP were detected in 9/12 tumours. VIP receptor mRNA was expressed in 5/12 tumours and functional VIP receptors were never demonstrated. The tumours having the highest VIP levels also had the highest PACAP contents and were associated with a watery diarrhoea syndrome due to activation of intestinal VIP receptors. As PACAP recognizes the PACAP receptors and the VIP receptors with the same high affinity it may contribute to the syndrome and is a likely candidate for an autocrine control of neuroblastoma cell growth and differentiation.     

Redescription of Alcedinectes alcyon (Acari:Hypoderatidae) from the belted kingfisher (Aves:Coraciiformes; Alcedinidae)

OBJECTIVE: To assess whether pre-operative chemotherapy reduces operative morbidity in children with intravascular extension of renal tumours. PATIENTS AND METHODS: Thirty children with intravascular extension of their renal tumour, treated in 10 different centres in the UK, were reviewed retrospectively. RESULTS: Twenty-nine patients had nephroblastoma and one child had clear cell sarcoma (favourable histology in 23, unfavourable histology in six). Patients were classified into stage II (17 patients), stage III (three patients) and stage IV (10 patients). Ultrasonography had been performed in 29 patients and had correctly diagnosed intravascular extension in 11 (40%); computed tomography (CT) was accurate in 93% of patients. A pre-operative diagnosis was made accurately in 24 patients, with caval extension in 18 and atrial extension in six. Nine patients underwent primary surgery, whilst 21 had pre-operative chemotherapy followed by delayed nephrectomy. In the latter group, the intravascular thrombus diminished in 16 patients. Five patients died, one from tumour rupture and four from extensive or progressive tumour disease; the overall 2-year survival was 83%. Unfavourable histology did not adversely affect survival, and patients having pre-operative chemotherapy appeared to have a better outcome. CONCLUSION: CT remains the best imaging modality to assess intravascular tumour extension. Pre-operative chemotherapy is recommended for patients with intra-caval extension of tumour. Those with intra-atrial extension or with hepatic vein obstruction (Budd-Chiari syndrome) may require a cardiopulmonary bypass and primary surgery.     

Comparative genomic hybridization and telomerase activity analysis identify two biologically different groups of 4s neuroblastomas

Chromosomal aberrations of 20 stage 4s neuroblastomas were analysed by comparative genomic hybridization (CGH). In a subset of 13/20 tumours, telomerase activity was evaluated by the telomeric repeat amplification protocol (TRAP). The CGH data were compared with the CGH results of ten stage 1 and 2 (stage 1/2) and 22 stage 3 and 4 (stage 3/4) neuroblastomas. A total of 17/20 stage 4s neuroblastomas did not progress clinically, whereas tumour progression with lethal outcome occurred in 3/20 cases. The CGH data of clinically non-progressing stage 4s tumours revealed a high rate of whole-chromosome aberrations (73.4%) with an overrepresentation of mainly chromosomes 2, 6, 7, 12, 13, 17, 18 and an underrepresentation of mainly chromosomes 3, 4, 11, 14. MYCN amplification or 1p deletion was observed in only 1/27 or 2/17 clinically non-progressing stage 4s tumours respectively, whereas all three progressive stage 4s neuroblastomas showed MYCN amplification, 1p deletion and, in 2/3 cases, distal 17q gains. Except for one case, telomerase activity was not observed in non-progressing stage 4s neuroblastomas. In contrast, 4s tumours with lethal outcome revealed elevated telomerase activity levels. Our data suggest that stage 4s neuroblastomas belong to two biologically different groups, one of which displays the genetic features of localized stage 1/2 tumours, whereas the other mimics advanced stage 3/4 neuroblastomas.     

Human papillomavirus infection and esophageal cancer: a nationwide seroepidemiologic case-control study in Sweden

BACKGROUND: Despite being immunogenic, gastric cancers overcome antitumour immune responses by mechanisms that have yet to be fully elucidated. Fas ligand (FasL) is a molecule that induces Fas receptor mediated apoptosis of activated immunocytes, thereby mediating normal immune downregulatory roles including immune response termination, tolerance acquisition, and immune privilege. Colon cancer cell lines have previously been shown to express FasL and kill lymphoid cells by Fas mediated apoptosis in vitro. Many diverse tumours have since been found to express FasL suggesting that a "Fas counterattack" against antitumour immune effector cells may contribute to tumour immune escape. AIM: To ascertain if human gastric tumours express FasL in vivo, as a potential mediator of immune escape in stomach cancer. SPECIMENS: Thirty paraffin wax embedded human gastric adenocarcinomas. METHODS: FasL protein was detected in gastric tumours using immunohistochemistry; FasL mRNA was detected in the tumours using in situ hybridisation. Cell death was detected in situ in tumour infiltrating lymphocytes using terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). RESULTS: Prevalent expression of FasL was detected in all 30 resected gastric adenocarcinomas examined. In the tumours, FasL protein and mRNA were co-localised to neoplastic gastric epithelial cells, confirming expression by the tumour cells. FasL expression was independent of tumour stage, suggesting that it may be expressed throughout gastric cancer progression. TUNEL staining disclosed a high level of cell death among lymphocytes infiltrating FasL positive areas of tumour. CONCLUSIONS: Human gastric adenocarcinomas express the immune downregulatory molecule, FasL. The results suggest that FasL is a prevalent mediator of immune privilege in stomach cancer.     

Evidence for the continuity of early problem behaviors: application of a developmental model

Mitotic index > 6, proliferating cell nuclear antigen (PCNA) index > 5%, high tumour grade and absence of progesterone receptors (PR) are significant predictors for poor outcome in meningiomas. Since MIB-1 (Ki-67) is a more specific cell proliferation marker, and overexpression of TGF-alpha is also associated with tumour progression, we compared the prognostic significance of these factors with the other indices. Intracranial meningiomas from 21 men and 36 women (age 54.5 +/- 1.7, mean +/- SEM) were classified as 24 benign, 24 atypical and nine malignant. Twenty-one of the 57 tumours recurred (mean interval to recurrence was 57.3 +/- 13.1 months). The mean follow-up period for patients without tumour recurrence was 81.9 +/- 8.7 months. MIB-1 labelling index (LI) was expressed as percentage of labelled nuclei to total tumour nuclei counted in the most densely labelled areas. Analysis of variance revealed significant differences between tumour grades for MIB-1 labelling indices (0.75 +/- 0.21 for benign, 3.2 +/- 0.57 for atypical 6.04 +/- 1.48 for malignant; P < or = 0.0066), and between malignant and non-malignant meningiomas for TGF alpha staining scores (P < or = 0.029). MIB-1 LI also correlated with mitotic and PCNA indices (P < or = 0.0001), but not with age of the patients. Male patients had higher tumour MIB-1 LI than females (P < or = 0.0128). Univariate analysis indicated that MIB-1 LI > 3%, TGF alpha score > 4 (scoring scale 0-5), mitotic index > 6, and negative PR status were significant factors for worse outcome. Higher MIB-1 LI, TGF alpha score and mitotic index as continuous variables were also significant negative predictors. With multivariate analysis, both MIB-1 LI and TGF alpha score remained significant factors when paired with all other variables: TGF alpha or MIB-1 LI, respectively, mitosis, PCNA, tumour grade, PR status, age, sex, postoperative radiation therapy. We conclude that MIB-1 LI and TGF alpha score are important independent prognostic indicators for patients with meningiomas.     

Androgen and oestrogen receptors in hepatocellular carcinoma and surrounding liver parenchyma: impact on intrahepatic recurrence after hepatic resection

Seventy-eight patients in whom androgen or oestrogen receptors, or both, were assayed in hepatocellular carcinoma (HCC) and the surrounding liver were discharged from hospital after curative resection of the tumour. Intrahepatic recurrence was evaluated retrospectively after 28-128 months follow-up to determine the association with receptor status. Androgen and oestrogen receptors in HCC significantly influenced the intrahepatic recurrence rate. The recurrence-free 5-year survival rate was 55 per cent for patients who had androgen receptor-negative tumours, 24 per cent for oestrogen receptor-negative, 10 per cent for oestrogen receptor-positive and 0 for androgen receptor-positive (P = 0.0322). Recurrence-free 5-year survival in 57 patients who had both receptor assays was 75 per cent for patients who had androgen receptor-negative, oestrogen receptor-negative tumours, 50 per cent for androgen receptor-negative, oestrogen receptor-positive, but 0 for androgen receptor-positive, oestrogen receptor-positive and androgen receptor-positive, oestrogen receptor-negative (P = 0.0104). The presence or absence of androgen or oestrogen receptor in the liver, however, was not associated with intrahepatic recurrence (P = 0.7534). Thus, androgen receptors are strongly associated with intrahepatic recurrence of HCC, while oestrogen receptors are weakly associated. Receptor status in the normal liver was not related to intrahepatic recurrence.     

Pharmacokinetics and pharmacodynamics of candesartan after administration of its pro-drug candesartan cilexetil in patients with mild to moderate essential hypertension--a population analysis

Among the peptide growth factors active in breast glandular cell proliferation epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are thought to play a major role in tumour development. They operate through binding to and activation of a common membrane receptor, defined as EGF-R. Their production is modulated by hormones and local growth factors. After it was shown by previous investigation in this laboratory that EGF-R could be detected in 90% of the tumours, but was masked by endogenous ligand in 36% of them, the question was raised as to the level of the ligand's expression in tumour tissue biopsies. Therefore, we investigated the expression of EGF and TGF alpha mRNA in 146 breast cancer biopsies by slot blot analysis using specific 32P-labelled probes. The data were correlated with sex steroids and EGF receptor content. Our results showed that EGF and TGF alpha coexisted in all tumour samples, and that their level of mRNA expression was similar in half of the tumours. Northern blot and polymerase chain reaction (PCR) analysis validated these findings. A significant direct correlation was found between the level of TGF alpha/EGF mRNA expression and the ER/progesterone receptor (PGR) content. TGF alpha and EGF mRNA levels were significantly higher in ER+ (P = 0.0015 and P = 0.0001, respectively) and in PGR+ tumours (P < 0.005 and P = 0.0001) than in their negative counterparts. Moreover, TGF alpha mRNA expression negatively correlated with the number of EGF-R binding sites measured by the standard method (P = 0.02), and it was significantly related to the number of sites occupied by endogenous ligand. In conclusion, it was shown that TGF alpha and EGF mRNA were coexpressed in all the tumour biopsies tested and that their level was higher in the hormone receptor positive than in negative samples. The correlation between the presence of ER/PGR sites, high level of TGF alpha/EGF mRNA and EGF-R occupancy by endogenous ligand is in favour of ER mediated control of TGF alpha and EGF production.     

A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurons after nerve injury

Several lines of evidence suggest that neurotrophin administration may be of some therapeutic benefit in the treatment of peripheral neuropathy. However, a third of sensory neurons do not express receptors for the neurotrophins. These neurons are of small diameter and can be identified by the binding of the lectin IB4 and the expression of the enzyme thiamine monophosphatase (TMP). Here we show that these neurons express the receptor components for glial-derived neurotrophic factor (GDNF) signaling (RET, GFRalpha-1, and GFRalpha-2). In lumbar dorsal root ganglia, virtually all IB4-labeled cells express RET mRNA, and the majority of these cells (79%) also express GFRalpha-1, GFRalpha-2, or GFRalpha-1 plus GFRalpha-2. GDNF, but not nerve growth factor (NGF), can prevent several axotomy-induced changes in these neurons, including the downregulation of IB4 binding, TMP activity, and somatostatin expression. GDNF also prevents the slowing of conduction velocity that normally occurs after axotomy in a population of small diameter DRG cells and the A-fiber sprouting into lamina II of the dorsal horn. GDNF therefore may be useful in the treatment of peripheral neuropathies and may protect peripheral neurons that are refractory to neurotrophin treatment.     

Evaluation of tryptamine in an impinger and on XAD-2 for the determination of hexamethylene-based isocyanates in spray-painting operations

We have investigated the relationship between immunohistochemically determined p53 status and outcome in 277 women with node-positive primary breast cancer who, following tumour excision and axillary clearance, were randomised to receive either 6 cycles of cyclophosphamide/methotrexate/S-fluorouracil (CMF) (n = 130) or no such post-operative treatment (n = 147). Follow-up data (median = 9 years) were available on all patients. A significant association was found between p53 status and survival. Patients with p53-positive tumours had a less favourable outcome than those with p53-negative disease. Women receiving adjuvant CMF chemotherapy had a significantly more favourable outcome compared to those who did not. The effect was seen both in women with p53-positive and p53-negative tumours; multivariate analysis showed relative risks for overall survival attributable to chemotherapy of 2.3 (95% CI 1.2-4.3) for women with p53-positive tumours and of 2.1 (95% CI 1.4-3.0) for those with p53-negative tumours. Thus, adjuvant chemotherapy with CMF is associated with a survival benefit in women with node-positive breast cancer irrespective of immunohistochemically determined p53 status.