Expression of vascular endothelial growth factor (VEGF) and its receptors in human neuroblastoma.

Angiogenic factors may play a role in the biology of neuroblastoma, a well vascularised tumour, which frequently spreads haematogenously. Therefore, we analysed expression of vascular endothelial growth factor (VEGF) in six human neuroblastoma cell lines and five primary neuroblastomas. High VEGF levels (1-3 ng/10(6) cells/day) were found in the supernatant of all cell lines examined (SK-N-LO, SK-N-SH, LS, SH-SY5Y, IMR-32, Kelly). VEGF peptide was also detected in tissue homogenates from four of five primary tumours. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that VEGF165 is the major isoform produced by neuroblastomas. In addition, all cell lines and primary tumours expressed the mitogenic VEGF receptor FLK-1, whilst the non-mitogenic receptor FLT-1 was less frequently positive, suggesting that the tyrosine kinase FLK-1 is involved in malignant transformation of neuroblastoma cells. However, neutralising antibodies to VEGF did not inhibit growth of neuroblastoma cell lines, which argues against a role of VEGF as an autocrine growth factor, at least for cell lines in vitro. We conclude that neuroblastoma cells produce VEGF, which may contribute to tumour vascularisation, growth and invasion.

Regression and progression in neuroblastoma. Does genetics predict tumour behaviour?

Neuroblastoma (NB) is a heterogeneous disease. The clinical course may range from spontaneous regression and maturation to very aggressive behaviour. Stage 4s is a unique subcategory of NB, generally associated with good prognosis, despite skin and/or liver involvement and the frequent presence of tumour cells in the bone marrow. Another type of NB is the locally invasive tumour without bone and bone marrow involvement which can also have a good prognosis, irrespective of lymph node involvement. Unfortunately, there is only limited biological information on such tumours which have not been treated with cytotoxic therapy despite lymph node involvement, residual tumour mass after surgery and/or bone marrow infiltration. In order to find specific genetic changes common to NBs with a benign clinical course, we studied the genetic abnormalities of these tumours and compared them with highly aggressive tumours. We analysed a series of 54 localised and stage 4s tumours by means of in situ hybridisation performed on fresh cells or on paraffin embedded tissues. In addition, we performed classical cytogenetics, Southern blotting and PCR analysis on fresh tumour tissue. The majority of patients had been treated with surgery alone, and in a number of patients tumour resection was incomplete. Deletions at 1p36 and amplifications of the MYCN oncogene were absent, and diploidy or tetraploidy were not seen in any case, with residual localised tumours possessing a favourable outcome. Unexpectedly, one patient with a tetraploid 4s tumour without any genetic structural changes not receiving any cytotoxic treatment, did well. Interestingly, this genetic spectrum contrasted with that of progressing tumours, in which most had genetic aberrations, the deletion at 1p36 being the most common event. These data, although limited, suggest that an intact 1p36 (recognised by D1Z2), the absence of MYCN amplification and near-triploidy (at least in localised tumours), represent prerequisites for spontaneous regression and/or maturation.

First experience with prognostic factors in unselected neuroblastoma patients. The Austrian Neuroblastoma 87 Study.

Between January 1987 and December 1993, 117 patients were registered in the Austrian A-NB87 study. The male/female ratio was 1.18, with 50 patients below the age of 1 year at diagnosis. Patients were assigned to stage according to the result of primary surgery in localised disease. Age, ferritin and neuron specific enolase were used in addition in stage III disease for risk-adapted treatment. Adrenal or pelvic primary tumour sites were mainly associated (81%) with advanced disease. The median observation time of the study is 3.5 years. The overall survival at 3 years was excellent in low stage disease and IVs patients, i.e. 100% for stage I and IIA (20 patients), 92% in stage IVs (13 patients), 81% in stage IIIA (18 patients) and 69% in stage IIB (8 patients). Stage IV (38 patients) showed a survival rate of 51%, whereas stage IIB (10 patients) had the worst outcome in this study, i.e. 20%, due to treatment-related toxicity. Significant unfavourable prognostic factors were neuron specific enolase (NSE) > 100 ng/ml, ferritin > 300 micrograms/ml and amplified MYCN. This study achieved a better survival rate in stage IV patients and a subgroup of stage III in comparison to our previous study (Pädiatrie und Pädologie 1986, 21, 269) and gives the basis to further reduce treatment intensity in low-risk disease based on biological factors. However, prognosis for high-risk cases was still poor in spite of a very aggressive treatment concept.

Requirement of residual thymus to restore normal T-cell subsets after human allogeneic bone marrow transplantation.

BACKGROUND: To determine the effect of residual thymic activity in reconstituting the T-cell system after T cell-depleting therapy, we monitored T-cell subsets of a unique thymectomized cancer patient in comparison to thymus-bearing patients after allogeneic bone marrow transplantation (BMT). METHODS: T cells and T-cell subsets previously shown in murine studies to be regulated by the thymus were analyzed by FACS from 6 to >48 months after BMT. The investigation of thymus-bearing patients included 32 examinations of 9 children and 14 adults. None of the investigated cases had severe graft-versus-host disease or severe infections when examined. RESULTS: In the thymectomized host, T-cell regeneration occurred by donor cell expansion and was characterized by two prominent features: (i) a persistent failure to regenerate naive (CD45RA+) T-helper cells (14%, median), consistent with the recently developed concept of a thymus-dependency; and (ii) persistently elevated proportions of CD3+CD4-CD8- cells (double-negative cells, median 29%), which were identified in T cell receptor (TCR)gamma delta+ (22%, median of CD3+ cells, 88% double negatives) but also TCRalpha beta+ T-cell populations (78%, median of CD3+ cells, 17% double negatives). In thymus-bearing patients, 10 of 12 and 6 of 14 examinations of children and adults, respectively, performed later than 12 months after BMT showed the proportion of CD4+CD45RA+ cells appropriate for age (>52% and >28% in children and adults, respectively). Elevated double-negative cells (>10%) were found in only three patients, but none had elevated double-negative cells with a TCRalpha beta+ phenotype. CONCLUSION: Residual thymic activity might, in addition to its well-established role for regenerating naive T-helper (CD4+CD45RA+) cells, control the expansion of double-negative cells. A normal T-cell subset regeneration in a proportion of thymus-bearing adult hosts indicates the potential of an effective residual thymic activity even beyond childhood.

Double high-dose chemotherapy with autologous peripheral stem cell rescue in relapsed Wilms' tumor.

We performed double high-dose chemotherapy (HDCT) and autologous stem cell rescue (ASCR) on an 8-year-old girl after relapse of Wilms' tumor with unfavorable histology. Relapse occurred while the patient was still on first-line therapy and showed multiple adverse prognostic features. Since depleted hematopoietic reserves restricted further conventional therapy, a double HDCT approach was chosen to achieve maximal dose intensity. Four years after relapse the girl is in second remission and well. Double HDCT with ASCR may represent a feasible approach to rescue extensively pretreated patients with recurrent Wilms' tumor of unfavorable histology.

CD34+-selected autologous peripheral blood stem cell transplantation (PBSCT) in patients with poor-risk hematological malignancies and solid tumors. A single-centre experience.

Between July 1994 and December 1996, PBSC were mobilized in 28 patients with poor-risk hematological malignancies and solid tumors. CD34+ cells were positively immunoselected using the Ceprate CS System. By December 1996, 22 patients had been reinfused with a median of 3.325 (0.078-9.5) x 10(6)/kg CD34+ cells. In three patients unselected back-up PBSC had to be transfused along with selected CD34+ cells because of a CD34+ cell number <0.5 x 10(6)/kg. G-CSF (10 microg/kg) was started on day +1 and all patients engrafted within a median day number of 12 (range, 10-22) until leukocytes >1.0 x 10(9)/l and a median day number of 56 (range, 10-180) until platelets >20.0 x 10(9)/l (ie platelet transfusion independence). Time to leukocyte and platelet recovery was significantly shorter in patients receiving >2.0 x 10(6)/kg purified CD34+ cells as compared to patients reinfused with <2.0 x 10(6)/kg CD34+ cells. The hematopoietic recovery time was similar to that of 18 historical control patients treated with unseparated ABMT +/- PBSCT with the exception of a significantly faster leukocyte engraftment in patients receiving >2.0 x 10(6)/kg CD34+ cells and a significantly delayed platelet recovery time in patients receiving <2.0 x 10(6)/kg purified CD34+ cells. There was a trend for a better overall survival and a lower probability of progression/relapse as compared to the historical controls. We observed five episodes of serious opportunistic infections (three pulmonary fungal infections, two cases of cryptosporidiosis) after the take. Four of these patients had been reinfused with <2.0 x 10(6)/kg CD34+ cells probably indicating a delayed immune reconstitution after CD34+-selected PBSCT.

Treatment of infant leukemia with busulfan, cyclophosphamide +/- etoposide and bone marrow transplantation.

Infants with acute leukemia have a poor chance of being cured by conventional chemotherapy. We therefore treated cases of infant leukemia with high dose chemotherapy followed by bone marrow transplantation (BMT). Six suffered from acute leukemia and one from refractory anemia with excess of blasts (RAEB-t). The conditioning regimen consisted of busulfan (BU) and cyclophosphamide (CY), and was intensified by adding etoposide (VP) in four cases. At the time of BMT the children were 4, 5, 12, 13, 13, 14, and 20 months old. Three children were autografted, three received HLA-identical marrow from a sibling donor, and one child received matched unrelated donor marrow. All five children who were grafted in complete (CR) or partial remission (PR) are alive and well in CR 7, 13, 24, 37, and 46 months after allogeneic (two patients) or autologous (three patients) BMT, and 13, 17, 29, 42, and 53 months after initial diagnosis. The child with RAEB-t and the one transplanted in second chemotherapy-resistant relapse of acute non-lymphoblastic leukemia relapsed at 7 and 17 months respectively. The chemotherapy regimen was well tolerated. BU-CY-VP is a promising alternative treatment to regimens including total body irradiation for very young children suffering from acute leukemia.

Peripheral blood stem cell (PBSC) collection in extremely low-weight infants.

While PBSC collection has become a safe procedure for adults, only a few reports exist about its efficacy, safety and feasibility in paediatric patients, especially extremely low-weight infants. We describe successful PBSC collection in three infants of less than 10 kg body weight (BW; range: 6.92-9.4 kg) suffering from stage IV neuroblastoma. Harvest of PBSC started after mobilisation with high-dose chemotherapy and G-CSF, as soon as 1.0% CD34+ cells were detected. Collections were performed using a Baxter CS-3000 Plus separator primed with a mixture of irradiated, white cell-depleted and CMV-negative packed red cells resuspended in 5% human albumin and diluted with saline to match the patient's haematocrit. Performing a median of four, (4-7, median, range) procedures we collected at least 4 x 10(8)/kg BW nucleated cells (NC) from all three patients. The infants were not sedated and showed no serious side-effects. All three children were successfully transplanted with myeloid engraftment in 8 (7-9) days, independence from red cell support was achieved in 15 (10-20) days and from platelet transfusions in 25 (14-29) days after PBSC infusion. We conclude that PBSC harvesting using continuous flow cell separators is safe, even in low-weight infants of less than 7 kg BW.

Effective T cell regeneration following high-dose chemotherapy rescued with CD34+ cell enriched peripheral blood progenitor cells in children.

The ex vivo enrichment for the CD34+ cell fraction of PBPC, while it retains the capacity to restore haematopoiesis and potentially reduces a contamination by tumour cells, implements a depletion of T cells. To test whether such a setting adversely affects T cell reconstitution, we monitored T cells in four paediatric patients after CD34+ selected PBPC transplantation. The dose of CD34+ cells, which were enriched to 74%, median, was 7.1 x 10(6)/kg, median, that of T cells was 0.071 x 10(6)/kg, median. The patients were homogenous with respect to features with a potential to effect T cell reconstitution (low median age, (35 years); stage IV malignant tumours in first CR, uncomplicated post-treatment course). The results of sequential FACS analyses showed that by 9 months after treatment all four patients had recovered (1) a normal T cell count (CD3+ cells 1434/microl, median); (2) a normal CD4+ cell count (816/microl, median), while CD8+ cells were recovered (>330/microl) already by 3 months; (3) a normal CD4/CD8 ratio (1.8, median), as a result of an augmented growth of CD4+ cells between 3 and 6 months (increase of CD4+ cells 4.9-fold, median, CD8+ cells 1.1-fold, median). Expansion of cells with a CD45RA+ phenotype (thymus-derived T cells) predominated; from 3 to 6 months the increase of CD4+/CD45RA+ T cells was 130-fold, that of CD4+/CD45RO+ cells was 1.7-fold; CD8+/CD45RA+ cells increased 9-fold, CD8+/CD45RO+ cells increased 2.1-fold, indicating effective thymopoiesis. The findings demonstrate that in paediatric patients the setting of HD-CTX rescued with autologous CD34+ selected PBPC per se is not predictive of an impaired T cell recovery. High thymic activity may be a key factor for the rapid restoration of T cells.

Isochromosome 12p and maternal loss of 1p36 in a pediatric testicular germ cell tumor.

Analysis of a pediatric germ cell tumor by conventional cytogenetic investigation and fluorescence in situ hybridization showed consistently the presence of two isochromosomes 12p, loss of the maternal band 1p36, and other numerical and structural chromosome changes. The rearrangements observed resulted mainly from breaks occurring at paracentromeric regions. This report represents the first description of i(12)(p10) in a pediatric testicular embryonal carcinoma.

NADH in the pyramidal cell layer of hippocampal regions CA1 and CA3 upon selective inhibition and uncoupling of oxidative phosphorylation.

N2-laser-induced fluorescence in combination with the time and spectral resolution of fluorescent NADH molecules allows on-line measurement of relative NADH concentration with high spatial resolution (diameter of optical fibre 200 microns, lambda(exc) = 337 nm, lambda(det) = 460 nm). Energy metabolism was impaired in submerged rat hippocampal slices using the inhibitors amytal, 3-nitropropionate (3-np), sodium cyanide (1 mM each) and the uncoupling agent 2,4-DNP (200 microM). A microprocessor-controlled repeated positioning of the optical fibre in CA1 and CA3 pyramidal cell layers, and CA1 stratum radiatum (CA1SR). Time-dependently, NADH fluorescence increased reversibly upon perfusion with amytal and cyanide. It was unchanged by perfusion with 3-np for 40 min and rapidly decreased upon perfusion with 2,4-DNP. The CA1/CA3 ratio of NADH fluorescence mildly decreased to 0.92 +/- 0.04 (mean +/- S.D.) at 10 min (P < 0.05) and 0.89 +/- 0.05 at 20 min (P < 0.01) upon perfusion with amytal. The CA1/CA3 ratio increased to 1.56 +/- 0.28 at 10 min (P < 0.01) and 1.29 +/- 0.35 at 20 min (P < 0.05) upon application of 2,4-DNP. Fluorescence in CA1SR was similar to fluorescence in CA1 upon perfusion with 2,4-DNP and similar to CA3 upon perfusion with amytal. We conclude that NADH fluorescence can be measured with high regional selectivity and specificity in hippocampal slices. Selective inhibition of mitochondrial complex I and uncoupling of energy metabolism differentially impair NADH concentration in different hippocampal areas.

Antiproliferative activity and apoptosis induced by retinoic acid receptor-gamma selectively binding retinoids in neuroblastoma.

Retinoids modulate several cell functions and especially inhibit the growth of tumor cells. Their biological activity is mediated by retinoic acid receptors (RARs), of which three subtypes (alpha, beta, gamma) have been identified. In human neuroblastoma (NB) reduced endogenous RAR-gamma expression was suggested to diminish the sensitivity for retinoids, to promote proliferation, and to contribute to the malignant phenotype. To correlate receptor selectivity with in vitro activity, we analysed the effect of six synthetic retinoids with selectivity for human RAR-alpha/beta/gamma on the human LAN-5 NB cell line and compared it with the natural compound all-trans-retinoic acid (ATRA). Apoptosis was determined by flow-cytometry using terminal-deoxynucleotidyl transferase to end-label DNA fragments in situ in apoptotic cells. The antagonist for RAR-beta/gamma CD2665 as well as the selective agonists for RAR-alpha CD336 and RAR-beta CD2019 were less effective in growth inhibition than ATRA. In contrast, the synthetic RAR-gamma selective agonists CD437 and CD2325 induced a concentration- and time-dependent antiproliferative effect, which was similar or even more pronounced than ATRA. In contrast to ATRA, the adition of CD437 and CD2325 did not induce morphological changes typical of NB cell maturation but resulted in morphological features consistent with the occurrence of programmed cell death. Flow-cytometric analysis showed that in contrast to ATRA the addition of CD 437 and CD 2325 results in progressive time-dependent increase of apoptotic cells (25.9% and 57.7% after 72 hours). In conclusion, our study demonstrates RAR-gamma selectively binding retinoids dramatically suppress NB cell growth, primarily by inducing programmed cell death rather than by cell differentiation. Since advanced or disseminated NB tumors endogenously express low levels of RAR-gamma and lack of apoptosis is involved in tumor progression, RAR-gamma selectively binding retinoids may be more appropriate retinoids for clinical trials in NB.

Long term remission of intrinsic brainstem gliomas: the case reports of two children.

Intrinsic brainstem gliomas carry the worst prognosis of all pediatric CNS tumors; only 10-25% of patients are expected to survive for more than two years. Over a period of four years seven intrinsic brainstem gliomas were diagnosed in children in one institution. Four of them underwent a rapidly fatal course, whilst one was diagnosed only two years ago, which is too recent for long-term evaluation. We report the case histories of the remaining two boys, who showed a favorable course of their disease. Presenting symptoms were headaches and signs of brainstem dysfunction with multiple bilateral cranial nerve palsies, ataxia and pyramidal tract signs. Diagnosis rested on neuroimaging (CAT scans and/or MRI scans). Both tumors were intrinsic brainstem gliomas, one diffuse and the other focal. They responded to treatment (radiotherapy and chemotherapy in the former patient and radiotherapy alone in the latter patient). The two boys became long-term survivors and have remained well, without evidence of disease, for more than 71 and 61 months, respectively, after completion of treatment. They are probably cured. Prompt therapy with curative intention is recommended, with consistent adherence to the chosen antitumor regimen even in poor-risk brainstem gliomas.

[Selection of physiologic bacterial flora by chemotherapeutic agents]. / Selektionierung der physiologischen Keimflora durch Chemotherapeutika.

A variety of antimicrobial agents has been shown to induce alterations in the bacterial homeostasis of the human microflora. Although the role of the normal flora is still poorly understood, there is evidence that alterations in the flora have a number of important clinical consequences. The normal flora acts as a natural defence against colonization or infection with pathogens. In addition, the altered flora may assume importance as a reservoir of potential pathogens. Thus, antibiotic-induced colonization predisposes patients to subsequent endogenous infections with these organisms which, in turn, have been rendered partially or totally resistant to formerly highly active agents. Broad spectrum antimicrobials with a high degree of biliary elimination show a marked impact on the faecal flora. Susceptible enteric bacteria are eliminated within 48 hours and are replaced by enterococci and Candida albicans. Recolonization occurs after discontinuation of therapy by multiresistant organisms like Klebsiella/Enterobacter and Pseudomonas. Oral antibiotics also lead to substantial alterations in the composition and resistance patterns of the faecal flora. In clinical medicine we should be aware of the substantial alterations of the human microflora which may accompany the use of antimicrobial agents.

Transplantation of related and unrelated umbilical cord blood stem cells in Austria. Austrian Working Party for Stem Cell Transplantation. Austrian Society of Hematology and Oncology.

Allogeneic bone marrow transplantation is limited by the availability of suitable HLA-matched donors and the risk of graft versus host disease (GvHD). In an attempt to overcome these limitations umbilical cord blood (UCB), has become a further alternative. UCB transplantations in Austria were started in 1991. As of September 31, 1998, six patients have been transplanted. Diagnoses were severe aplastic anaemia (SAA) (n = 2), acute lymphoblastic leukaemia (ALL) (n = 1), familial hemophagocytic syndrome (FHL) (n = 2) and chronic myelomonocytic leukaemia (CMML) (n = 1). Three patients received UCB grafts from HLA-identical siblings and three patients from unrelated donors, of whom two were disparate at two HLA loci (A/B) and one mismatched at one locus (C). Five patients were engrafted with complete donor hematopoiesis, with a median time of 26.5 days (range 14 to 39 days) to an ANC count of > or = 0.5 x 10(9)/L and a median time of 42.5 days (range 24 to 67 days) to a platelet count of > or = 20 x 10(9)/L. One patient with FHL had partial engraftment and died due to reactivation of cytomegalovirus (CMV) infection and CMV pneumonia on day +25. Of the five patients surviving the post-transplant period, one with CMML had a relapse on day +128 and died after a HLA-matched bone marrow transplantation from the same sibling donor in the second relapse. Another patient with ALL relapsed on day +200 but is still alive under palliative treatment; one patient with SAA showed graft rejection and autologous hematopoietic reconstitution and later had a successful CD34(+)-selected allogeneic peripheral stem cell transplant from a C-locus mismatched unrelated donor. Two patients (one with SAA and one with FHL) are alive with complete remission of the underlying disease. This report reflects the experience and results of UCB transplantation in Austria and discusses the position of UCB transplantation in the context of the other stem cell alternatives available today.

The tongue, the lingometer, and the role of accommodation in occlusion.

A new diagnostic swallowing device (lingometer) and its use to detect deforming tongue movements is described. A theory explaining the relationship of these movements to arch formation is also presented.

[Diagnostic value of sonography and double contrast arthrography in meniscus lesions]. / Diagnostische Wertigkeit von Sonographie und Doppelkontrastarthrographie bei Meniskusläsionen.

Using a real time scanner and a 7.5 mHz transducer, the meniscus can bei visualised as a homogeneous triangle. It is clearly distinguishable from the tibial plane und the condyle of the femur. Tears in the meniscus show up as a double contour rich in echoes with an intervening low-echo area, or as a contour rich in echoes with an adjacent low-echo area. The posterior horn area can be visualised most clearly. Assessment of the interior part of the meniscus is somewhat easier than visualising the exterior part. In a clinical study with 107 menisci examined by sonography and controlled by arthroscopy or arthrotomy the rate of accurate diagnoses by sonography of the meniscus is 82%. 42 of these menisci were additionally examined by double contrast arthrography besides sonography. This yielded an accuracy rate of 74% for double contrast arthrography; the latter is superior to sonography only in the anterior horn area. Problems in respect of meniscus sonography occur only in case of transverse ruptures, scars and longitudinal meniscus ruptures presenting as bucket handle tears near the base. The typical longitudinal tear in the area of the posterior horn can be visualised most clearly. Analysis of the results shows that sonography of the meniscus is a noninvasive, painless and randomly reproducible and risk-free examination method which has a diagnostic value especially in the area of the posterior horn that can be compared with double contrast arthrography. Further studies must show whether with an increasing spread of the method it would be possible to replace double contrast arthrography by sonography in diagnosis of menisceal trouble.