MYCN protein expression as a predictor of neuroblastoma prognosis

About half of nonlocalized neuroblastomas have MYCN gene amplification and usually progress rapidly, but the half without such amplification also do poorly, albeit progressing more slowly. We hypothesize that overexpression of MYCN protein can occur without gene amplification and that this expression reliably predicts the prognosis of neuroblastoma. To determine whether MYCN expression correlated with outcome, we assayed MYCN protein immunohistochemically in 180 archival pretreatment and posttreatment samples and stratified the 57 conventionally treated stage IVS, III, and IV patients by these conventional prognostic factors: stage, age, serum ferritin, Shimada histology, urinary catecholamine ratio, and MYCN gene status. At a median follow-up of >/=6.8 years, we found in patients with known MYCN gene status that the 23 of 37 without gene amplification fared no better than the 14 of 37 with gene amplification (P = 0.35 and 0.21, comparing relapse-free and survival rates). Conversely, in patients without MYCN gene amplification, 9 of 23 were found to overexpress MYCN protein pretreatment, and they did worse than the 14 of 23 without detectable MYCN protein (P = 0.0016 and 0.022, comparing relapse-free and survival rates). Furthermore, MYCN protein expression was prognostic without (P = 0.00001) and with (P = 0.0007) stratifying all 57 patients by MYCN gene status, each conventional prognostic factor (P ranging from 0.00001-0.013), or simultaneously by the two most important factors, stage and age (P = 0.00076). We conclude that overexpression of MYCN protein without gene amplification correlated significantly with the clinical behavior of neuroblastoma and predicted outcome independently of other prognostic factors. This strongly supports the hypothesis that expression of the MYCN oncogene is critical for progression of neuroblastoma.     

CD44H expression by human neuroblastoma cells: relation to MYCN amplification and lineage differentiation

The human CD44 cell surface glycoprotein has been involved in a variety of functions including lymphocyte homing, extracellular cell matrix attachment, and tumor metastasis. Due to the alternative splicing of the single gene, a large family of different variants or isoforms is generated. Several reports have indicated an up-regulation of CD44 variant (v) isoforms in malignant process, conferring metastatic potential to non-metastatic cells. Neuroblastoma is a tumor characterized by an aggressive and metastatic behavior in advanced stages with amplification of the MYCN protooncogene. In this report we show that the CD44 standard molecule is highly expressed in 100% of stage I-III, IVs neuroblastomas and ganglioneuromas but only in a subset of stage IV tumors. In contrast, no expression of CD44 was detected on MYCN amplified stage IV tumors, thus demonstrating a highly significant negative relationship between MYCN amplification and CD44 expression in neuroblastoma. The expression of CD44 on neuroblastoma cultured cell lines was not shown to be related to MYCN amplification but rather linked to the S-type, schwann/glial differentiation lineage. Immunochemical analysis of tumor samples with anti-CD44v3 and -v6 antibodies and Northern blot analysis of mRNA from cell lines with probes spanning exons 4-10 did not reveal any expression of splice variants on neuroblastomas of all stages and cell lines, thus ruling out a major role of these isoforms in neuroblastoma progression and metastasis.     

Activity of a 40 kDa RNA-binding protein correlates with MYCN and c-fos mRNA stability in human neuroblastoma

Subclones of neuroblastic (N) and substrate adherent (S) cells have been established from neuroblastoma tumours cultured in vitro which differ in growth characteristics and MYCN expression. N cells derived from the NBL-W cell line (W-N) express 5-fold higher levels of MYCN mRNA and 10-fold higher levels of MYCN protein than S cells (W-S), despite having the same MYCN copy number. In an effort to identify the molecular mechanisms responsible for the disparity in steady-state MYCN levels, the rate of MYCN mRNA degradation was measured in the two subclones. The half-life of MYCN mRNA in the W-N cells was approximately 45 min compared to approximately 6 min in the W-S cells. Similarly, the half-life of another labile mRNA, c-fos, differed in W-N and W-S cells (30 min versus 15 min, respectively). The turnover of labile mRNAs is thought to be mediated by the interactions of trans-acting factors with AU-rich elements within the 3' untranslated region. RNA UV cross-linking assays using W-N cell lysate demonstrated abundant quantities of a protein, 40 kDa in size (p40), that bound specifically to AU-rich elements within the MYCN and c-fos 3' untranslated region. However, p40 was barely detectable in W-S cells. Our studies suggest that p40 may play a role in determining neuroblastoma phenotype by regulating MYCN and c-fos mRNA turnover.     

Evidence that the MYCN oncogene regulates MRP gene expression in neuroblastoma

We have recently shown that expression of the multidrug resistance-associated protein (MRP) gene is a powerful prognostic indicator in childhood neuroblastoma and have suggested that the MYCN oncogene may regulate MRP gene expression. To address this hypothesis, we have examined the relationship between MYCN and MRP gene expression in neuroblastoma tumours and cell lines. MYCN and MRP gene expression were highly correlated in 60 primary untreated tumours both with (P = 0.01) and without MYCN gene amplification (P < 0.0001). Like MRP, high MYCN gene expression was significantly associated with reduced survival, both in the overall study population and in older children without MYCN gene amplification (relative hazards = 13.33 and 19.61, respectively). Inhibition of MYCN, through the introduction of MYCN antisense RNA constructs into human neuroblastoma cells in vitro, resulted in decreased MRP gene expression, determined both by RNA-PCR and Western analysis. The data are consistent with MYCN influencing neuroblastoma outcome by regulating MRP gene expression.     

Fluorescence in situ hybridization analysis of chromosome 1p36 deletions in human MYCN amplified neuroblastoma

Artificial illumination is an important factor in the management of layers. In this study, a new monochromatic light system was developed for egg layers. Prelaying pullets (Lohmann) were marked and housed in nine light and temperature control rooms (15 battery cages, 3 hens per cage; n = 45), divided into three light treatments: 0.1 and 0.01 W/m2 light intensity using light emitting diode (LED) lamps and 0.1 W/m2 using mini-fluorescent bulbs (PL) (control). In each of the LED rooms, three wavelengths were tested: 560 (n = 9), 660 (n = 9), 880 (n = 6), and 660 intermitted lighting (15 min light 45 min dark, 660IN) (n = 9). Birds were exposed to 12 h light and 12 h of darkness using PL lamps. At 21 wk of age, the light period was increased to 12.75 h by using 5.5 h of LED lamps and 7.25 of PL light source for Groups 1 and 2, the third group received 12.75 h of PL light. Until 28 wk of age, light hours increased by 0.5 h/w using LED light for Groups 1 and 2 and PL source for the third group, reaching 16 h of light at 28 wk of age. Egg production and feed consumption were recorded daily; egg components were recorded weekly for 10 mo. A significant reduction in egg production was observed in all 880nm groups; no differences in egg production and quality were found in the other groups. Feed consumption was significantly lower by 7% in all 0.01 W/m2 groups. We suggest that an important reduction in rearing costs of laying hens may be obtained by using this system.     

Intractable diarrhea in a boy with vasoactive intestinal peptide-producing ganglioneuroblastoma

A 1-year-old boy had intractable diarrhea and symptoms of the watery-diarrhea-hypokalemia-achlorhydria (WDHA) syndrome, a well-known entity in adults. Resection of a ganglioneuroblastoma situated in the neck caused prompt relief of symptoms. The ganglioneuroblastoma in this instance contained the enterohormone vasoactive intestinal peptide (VIP); blood levels of this peptide were elevated preoperatively. After tumor resection, the VIP level returned to normal, and the diarrhea ceased on the day of the operation. The genesis of the diarrhea in relation to the production of polypeptides from neuroendocrine origin is discussed (APUD-cell concept). VIP may be the mediator of the WHDA syndrome in ganglioneuroblastoma.     

Hypocomplementemic membranoproliferative glomerulonephritis in a child with ulcerative colitis (author's transl)

A 5 1/2 year old girl with hypocomplementemic membranoproliferative glomerulonephritis suffered from severe nephrotic syndrome. Despite intensive treatment with corticosteroids and immunosuppressive drugs the clinical state deteriorated. Three years after clinical onset of the disease the girl entered our regular hemodialysis program because of terminal renal insufficiency. After two weeks of intermittent hemodialysis she presented intestinal bleeding, which could not be stopped. One week later complete ileus developed and the child died. Before the onset of melaena no occult blood or mucus could be detected in the faeces. The autopsy revealed a severe ulcerative colitis with pseudopolyposis of the whole colon. In serum specimens still available colonic antigen could be detected by means of immunodiffusion using a rabbit antiserum against fetal colonic extract. Immunofluorescence studies showed granular deposits of immunoglobulins and complement along the glomerular capillary walls suggesting an immunogenesis of the glomerulonephritis by circulating immune complexes. The possibility of an interrelationship in the pathogenesis of both diseases is discussed. It should not be excluded that immune complexes formed in excess of colonic antigen have caused or perpetuated chronic glomerulonephritis.     

Acute diarrhea as a leading symptom of ganglioneuroblastoma

Although prophylaxis with antibiotics cannot avoid all cases of infectious endocarditis, it remains the main approach capable of reducing the frequency of this severe disease. It consists in using an adapted antibiotic cover in situations exposing to bacteraemia in patients at-risk with known cardiopathy. In France, recommendations have been established by a consensus conference in 1992. The fact that the incidence of this infection has not decreased merits recalling these recommendations.     

Pediatric malignant glioma with tubuloreticular inclusions and MYCN amplification. Report of a case with immunohistochemical, ultrastructural, flow cytometric, karyotypic, and Southern blot analysis

BACKGROUND: The authors described unusual pathologic features in a left frontal lobe malignant glioma in a 31/2-year-old boy. The pathology was similar in the initial excision and two subsequent recurrences at 9 and 11 months and at autopsy, when extensive subarachnoid spread was noted. METHODS: The tumor was studied by conventional histology, immunohistochemistry, flow cytometry, transmission electron microscopy (TEM), immune electron microscopy (IEM), and cytogenetic and Southern blot analysis. RESULTS: The tumor revealed two different histologic patterns. One component showed large cells with eosinophilic cytoplasm, vesicular nuclei with prominent nucleoli, eosinophilic perinuclear inclusions, and immunoreactivity for glial fibrillary acidic protein (GFAP) and vimentin. The other component consisted of undifferentiated cells with hyperchromatic nuclei and scanty cytoplasm. By TEM, the perinuclear aggregates were composed of tubuloreticular inclusions, which were also observed in endothelial cells within the tumor vasculature. By IEM, the intermediate filaments in the tumor cell cytoplasm were decorated with GFAP. Flow cytometric results revealed a marked increase in the S-phase (48%), whereas cytogenetic analysis of short-term cultures showed an abnormal karyotype containing marker chromosomes and double minutes. In the second resection, additional karyotypic abnormalities were noted, including 1p- and several additional markers. The first and second resections showed MYCN amplification by Southern Blot analysis in the 60- to 80-fold range. CONCLUSIONS: This tumor presents unique histologic, ultrastructural, and cytogenetic findings as well as MYCN amplification that is notable for a pediatric malignant glioma. Tubuloreticular inclusions were a prominent feature in this tumor, which again is unique for a glioma.     

Restitution of lunate bone necrosis in a child (author's transl)

We reported a case of aseptic bone-cartilage necrosis of the os lunatum in a child. All indications for constitutional disposition, anatomic variations in the wrist joint or a traumatizing of the wrist joint were absent. Etiologically, a disturbance in vascularization could be assumed. The increased repair potential in children apparently led to a restitution which has not been observed in adults.     

Extra-osseous localisation of 99Tcm methylene diphosphonic acid (MDP) in a primary neuroblastoma

A case of primary neuroblastoma in a seven year old boy is presented in which 99Tcm methylene diphosphonic acid (MDP) used as a bone scanning agent localised in the primary tumour. The possible mechanism for this is discussed.     

c-MYC amplification in a case of progression from MDS to AML (M2)

Serious side effects of photodynamic therapy (PDT) are rare (A.L. Abramson, M.J. Shikowitz, V.M. Mullooly, Clinical effects of photodynamic therapy on recurrent laryngeal papillomas, Arch. Otolaryngology Head Neck Surg., 118 (1992) 25-29; A.L. Abramson, M.J. Shikowitz, Clinical exacerbation of systemic lupus erythematosus after photodynamic therapy of laryngotracheal papillomatosis, Laser Surg. Med., 13 (1993) 677-679). The most frequent side effects of PDT are hypersensitive skin reactions, local edema, nausea, a metallic taste and liver-toxicity (J. Feyh, E. Kastenbauer, Treatment of laryngeal papillomatosis with photodynamic laser therapy, Laryngorhinootologie, 71 (1992) 190-192; J. Feyh, R. Gutmann, A. Leunig, Die Photodynamische Therapie im Bereich der Hals-, Nasen-, Ohrenheilkunde Laryngorhinootologie, 72(6) (1993) 273-78; M.S. Kavuru, A.C. Mekta, Treatment of recurrent respiratory papillomatosis, N. Engl. J. Med., 326 (1992) 204-205; B.L. Wenig, D.M. Kurtzmann, Photodynamic therapy in the treatment of squamous cell carcinomas of the head and neck, Arch. Otolaryngol Head Neck Surg., 116 (1990) 1267-1270). In this case a patient (aged 57 years) suffering from a recurrent larynx papillomatosis was treated with PDT. He was sensitized with Photosan 3 (DHE) 2.5 mg kg-1 body weight, 24 h prior to photoradiation. As a light source, an argon dye laser, operating at a wavelength of 630 nm was used, coupled with a cylindrical light applicator. After treatment the patient was admitted to an intensive care unit for 24 h. 3 h after photoradiation, general urticarial wheals arose, as well as tachycardia and a decrease in blood pressure followed by all the signs of serious anaphylaxis. 1.5 h after treatment with adrenaline and cortisone and stabilization of the cardiac and circulatory situation no more skin lesions were visible.     

Combined determination of N-myc oncogene amplification and DNA ploidy in neuroblastoma. Complementary prognostic indicators

BACKGROUND: N-myc gene amplification is a well-established prognostic indicator in neuroblastoma. Flow cytometric analysis of nuclear DNA content has shown that an abnormal nuclear DNA content in neuroblastoma is associated with a better prognosis. Because some patients with N-myc unamplified tumors have a poor prognosis, factors other than N-myc amplification may play a role in determining the clinical behavior of neuroblastoma. In the current study, the authors correlated N-myc gene amplification and flow cytometric nuclear DNA content with respect to prognosis. METHODS: Forty-one patients with neuroblastoma, including 15 screened patients, served as subjects. The copy number of the N-myc gene was determined by Southern blot analysis. DNA ploidy analysis was done on nuclei isolated from formalin-fixed, paraffin-embedded blocks. RESULTS: Of 40 specimens of neuroblastoma, 7 involved tumors containing amplification of the N-myc gene and 33 did not; 13 specimens showed DNA diploidy, and 27 showed DNA aneuploidy (including 4 with DNA tetraploidy). The Kaplan-Meier survival analysis indicated a significantly better prognosis in patients with unamplified N-myc tumors compared with those with N-myc amplified tumors (87.3% versus 28.6%, P < 0.05) and in patients with DNA aneuploid tumors compared with those with DNA diploid tumors (96.3% versus 43.0%, P < 0.001). The difference in the survival of the two extreme combinations, (e.g., 25 with N-myc unamplified and DNA aneuploidy [4 tetraploidy] versus 5 with N-myc amplified and DNA diploidy) was more significant (96.0% versus 20.0%, P < 0.001) than any other combination. CONCLUSION: Evaluations of N-myc gene amplification and DNA ploidy are complementary, and the combined determination of these two factors may be one of the most powerful prognostic indicators in neuroblastoma.     

Two novel mutations in the thyrotropin (TSH) receptor gene in a child with resistance to TSH

The TSH receptor is a G protein-coupled receptor that mediates the effects of TSH in thyroid development, growth, and synthetic function. We report here that a child with features of TSH resistance, including markedly increased serum TSH concentrations and low normal thyroid hormone levels, is a compound heterozygote for two novel mutations in the TSH receptor gene. One allele has a G to A transition corresponding to an arginine to glutamine change at codon 109 (R109Q) in the extracellular domain of the receptor. The other allele has a G to A transition corresponding to a premature termination codon at tryptophan 546 (W546X) in the fourth transmembrane segment. Each parent is heterozygous for one mutation, and both parents have normal thyroid function. Cells transiently transfected with the R109Q mutant exhibited reduced membrane binding of [125I]TSH and impaired signal transduction in response to TSH. In contrast, the W546X mutant was nonfunctional, with negligible membrane radioligand binding. Our findings indicate that a single normal TSH receptor allele is sufficient for normal thyroid function, but that the compound abnormality in the proband leads to TSH resistance.