Induction death and treatment-related mortality in first remission of children with acute lymphoblastic leukemia: a population-based analysis of the Austrian Berlin-Frankfurt-Münster study group.

In the management of the childhood acute lymphoblastic leukemia (ALL), 5% of failures are due to induction death and treatment-related deaths in first complete remission. We retrospectively analyzed the incidence, pattern and causes of death and its risk factors for 896 children with ALL enrolled into five Austrian (A) Berlin-Frankfurt-Münster (BFM) trials between 1981 and 1999. The estimated 10-year cumulative incidence of death significantly decreased from 6+/-1% (n=16/268) in trials ALL-BFM-A 81 and ALL-A 84 to 2+/-1% (n=15/628) in trials ALL-BFM-A 86, 90 and 95 (P=0.006). A significant reduction of death was evident during induction therapy (2.2% in trials ALL-BFM-A 81 and ALL-A 84 and 0.2% in trials ALL-BFM-A 86, 90 and 95, P=0.001). Of 31 patients, 21 (68%) patients died from infectious and 10 (32%) from noninfectious complications. Treatment in trial ALL-BFM-A 81, infant age and female gender were independent predictors of an enhanced risk for death. Conclusively, we found a progressive reduction of death rates that may be explained by the increasing experience in specialized hemato-oncologic centers and improved supportive and intensive care. We also identified a distinct subset of patients who are especially prone to death and may need a special focus when receiving intense chemotherapy.

ICA analysis of retina images for glaucoma classification.

Glaucoma represent one of the most frequent causes of partial loss of the visual field. It comes along with an ongoing destruction of the optic nerve caused by an increased pressure of the eye liquid. The disease becomes obvious from investigations of the retina with scanning laser microscopes. In this report an image analysis and classification system based on independent component analysis and k-nearest-neighbor classification is proposed. The method is tested with 120 selected retina images collected with the Heidelberg Retina Tomograph and achieves a classification rate of 91%.

Living at high altitude and risk of hospitalisation for atopic asthma in children: results from a large prospective birth-cohort study.

BACKGROUND: Asthma is among the most common chronic diseases in childhood and is steadily increasing in prevalence. Better characterisation of factors that determine the risk of hospitalisation for atopic asthma in childhood may help design prevention programmes and improve our understanding of disease pathobiology. This study will focus on the altitude of residence. METHODS: This is an ongoing prospective birth-cohort study that enrolled all live-born infants in the Tyrol. Between 1994 and 1999, baseline data were collected for 33 808 infants. From 2000 to 2005, all children hospitalised for atopic asthma at the age of > or =6 years (n = 305) were identified by a careful search of hospital databases. Disease status was ascertained from the typical medical history, a thorough examination and proof of atopy. RESULTS: Living at higher altitude was associated with an enhanced risk of hospitalisation for atopic asthma (multivariate RRs (95% confidence interval 2.08 (1.45 to 2.98) and 1.49 (1.05 to 2.11) for a comparison between altitude categories > or =1200 m and 900-1199 m versus <900 m; p<0.001). This finding applied equally to hospital admissions in spring, summer, autumn and winter. When altitude of residence was analysed as a continuous variable, the risk for asthma hospitalisation increased by 7% for each 100-m increase in altitude (p = 0.013). CONCLUSIONS: This large prospective study shows a significant association between the risk of hospitalisation for atopic asthma and altitude of residence between 450 and 1800 m. The underlying mechanisms remain to be elucidated, but it is tempting to speculate about a role for altitude characteristics such as the decline in outdoor temperature and air humidity and increase in ozone levels, which may trigger airway hyper-responsiveness and attenuate lung function.

High expression of the chemokine receptor CXCR4 predicts extramedullary organ infiltration in childhood acute lymphoblastic leukaemia.

Childhood acute lymphoblastic leukaemia (ALL) is a malignancy with the potential to infiltrate the liver, spleen, lymph nodes and brain. Such extramedullary presentation is important for understanding the biology of childhood ALL and also for developing new prognostic parameters. A potential mechanism in the trafficking of leukaemia cells is the interaction of the chemokine receptor CXCR4, which is expressed on ALL cells, and its ligand stromal cell-derived factor-1 (SDF-1), produced by stromal cells in bone marrow and extramedullary organs. Functionality of CXCR4 was demonstrated by a high correlation between cell surface density of CXCR4 and transendothelial migration of leukaemia blasts towards a gradient of SDF-1 (r = 0.73, P = 0.001). Inhibition of SDF-1-induced migration by an anti-CXCR4 monoclonal antibody (78.33 +/- 23.86% inhibition) evidenced the specificity of CXCR4 to SDF-1. In order to evaluate clinical significance of CXCR4 expression, lymphoblasts from the bone marrow of 73 patients with and without extramedullary organ infiltration were compared. Multiparameter flow cytometry revealed that lymphoblasts from patients with high extramedullary organ infiltration, defined as ultrasonographically measured enlargement of liver or spleen, expressed the CXCR4 receptor at higher fluorescence intensity (median 66.12 +/- 66.17) than patients without extramedullary organ infiltration (median 17.56 +/- 19.29; P < 0.001). Consequently, high expression of CXCR4 was strongly predictive for extramedullary organ involvement, independently of the peripheral lymphoblast count. Highest CXCR4 expression was seen in mature B ALL (median 102.74 +/- 92.13; P < 0.003), a disease characterized by a high incidence of extramedullary bulky disease. As high expression of the chemokine receptor CXCR4 predicts extramedullary organ infiltration in childhood ALL, we suggest that CXCR4 and its ligand play an essential role in extramedullary invasion.

Prognostic significance of DNA di-tetraploidy in neuroblastoma.

BACKGROUND: Identification of biological factors may provide tools to discriminate poor risk neuroblastoma patients of diagnosis, to ultimately offer risk adapted treatment intensity. PROCEDURES: Tumour cell DNA content, MYCN amplification (NMA), deletion of the short arm of chromosome 1 (del 1p) as well as three serological markers were assessed in 179 children with neuroblastoma. RESULTS: Localised regional disease (stage 1 to 3) was diagnosed in 98 patients, and disseminated disease in 81 patients (65 with stage 4, 16 with stage 4s). Median age at diagnosis was 12 months and the median observation time 4 years. Sixty-seven of 179 patients had near di-tetraploid tumours (37%), with a significantly worse prognosis of 44% overall survival at 4 years in comparison with 88% in near triploid tumours (P < .001). The near di-tetraploid group showed a significant correlation with additional adverse biological factors (NMA, del 1p: P < 0.001), age over 1 year (P< 0.001), clinical stage 4 (P< 0.001), elevated ferritin (P = 0.023), and elevated LDH (P< 0.001). Multivariate analysis based on the overall (OS) and event free survival (EFS) estimations revealed that near di-tetraploidy was the most powerful biological factor, with a P-value of <0.001 for EFS and OS, followed by NMA (P = 0.015) for OS and del 1p (P= 0.047) for EFS. CONCLUSIONS: This analysis underlines the important influence of near di-tetraploidy on prognosis, and suggests that more efforts should be undertaken to implement this factor in future studies.

Chromosomal regions involved in the pathogenesis of osteosarcomas.

The comparative genomic hybridization technique (CGH) was used to identify common chromosomal imbalances in osteosarcomas (OS), which frequently display complex karyotypic changes. We analyzed 13 high-grade primary tumors, 5 corresponding cell lines, 2 primary tumors grade 2, and 1 recurrent tumor from a total of 16 patients. Some of the CGH results have been verified by fluorescence in situ hybridization (FISH) studies. Gains of chromosomal material were more frequent than losses. Most common gains were observed at 8q (11 cases), 4q (9 cases), 7q (8 cases), 5p (7 cases), and 1p (8 cases). The smallest regions of overlap have been narrowed down to 8q23 (10 cases), 4q12-13 (8 cases), 5p13-14 (7 cases), 7q31-32 (7 cases), 8q21 (7 cases), and 4q28-31 (5 cases). These data demonstrate that a number of chromosomal regions and even two distinct loci on 4q and 8q are involved in the pathogenesis of OS, with gain of 4q12-13 chromosomal material representing a newly identified locus. Seven of 16 cases displayed, besides gain of 8q23 sequences, gain of MYC copies in CGH and FISH. Previous CGH reports confined gain of 8q material to 8cen-q13, 8q21.3-8q22, and 8q23-qter, whereas our data suggest that the loci 8q21 and 8q23-24 are affected in the development of OS. In contrast to recent reports, copy number increases at 8q and 1q21 did not have an unfavorable impact on prognosis in the present series. Genes Chromosomes Cancer 28:329-336, 2000.

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.

Epstein-Barr virus-negative boys with non-Hodgkin lymphoma are mutated in the SH2D1A gene, as are patients with X-linked lymphoproliferative disease (XLP).

X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency, which most often manifests itself after Epstein-Barr virus (EBV) infection. The main clinical phenotypes include fulminant or fatal infectious mononucleosis, dysgammaglobulinaemia and malignant lymphoma. We have recently cloned the SH2D1A gene, which has been shown to be mutated in approximately 70% of XLP patients. Now we report five novel SH2D1A mutations in patients from five unrelated XLP families. No mutations were found in another three XLP families. In three boys with early onset non-Hodgkin lymphoma (NHL) from two unrelated families a deletion of SH2D1A exon 1 and a splice site mutation were found, respectively. These patients did not show any laboratory or clinical signs of a previous EBV infection. A fourth EBV-uninfected and unrelated boy with a stop mutation in the SH2D1A gene shows only signs of dysgammaglobulinaemia. Development of dysgamma-globulinaemia and lymphoma without evidence of prior EBV infection in four of our patients suggests that EBV is unrelated to these phenotypes, in contrast to fulminant or fatal infectious mononucleosis. The role of SH2D1A as a putative tumour suppressor gene remains to be investigated.

Cryptic exons as a source of increased diversity of Ewing tumor-associated EWS-FLI1 chimeric products.

In the Ewing family of tumors (EFT), the EWS gene is rearranged with members of the ets oncogene family. Variability in genomic breakpoint locations is the source of significant heterogeneity in fusion product structure. As a consequence of variably included exon sequences from the two partner genes, a variable amount of additional peptide sequence is inserted in between the minimal transforming domains. Some of this molecular diversity has recently been correlated with disparate clinical outcome. Here we report on cryptic exons found in the chimeric RNA of three EFT with different EWS-FLI1 fusions. In two tumors, the emergence of a cryptic exon from FLI1 intron 5 in the chimeric RNA was apparently unrelated to the genomic rearrangement that occurred in FLI1 introns 4 and 5, respectively. In one case, a novel exon was generated through the creation of an artificial splice acceptor site in FLI1 intron 6 by the genomic rearrangement that occurred in EWS intron 8. These results further extend the spectrum of molecular diversity in EFT.

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.

Cortical NADH during pharmacological manipulations of the respiratory chain and spreading depression in vivo.

The nicotinamide adenine dinucleotide (NADH) is one of the main means for energy transfer in the mitochondrial respiratory chain and is an important parameter of cellular metabolism. NADH can be measured by its fluorescence and various fluorometric methods have been developed. In this study, a pulsed nitrogen laser combined with a fibreoptic set-up and photomultipliers was used to induce and measure NADH fluorescence on the cortical surface. The aim of the study was to assess the suitability of the laser induced spectroscopy for in vivo and on-line measurement of NADH in neuroscience and particularly for the assessment of neuronal metabolism. Changes in cerebral blood flow may affect fluorescence measurement. To assess the consequences of alterations in blood flow, the vasodilators glyceryl trinitrate and nimodipine and the vasoconstrictor endothelin-1 were applied. The induced hemodynamic changes were verified by colour Doppler sonography. The tests using the vasodilators showed that an increased blood flow in the brain increased not only NADH fluorescence but also the scattered light measured. The vasoconstrictor caused opposite effects. Insertion of a compensation method (subtraction of the scattered light) allowed the exclusion of hemodynamic artifacts. Effects of changes in the cellular metabolism were induced by sodium cyanide, an inhibitor of the mitochondrial respiratory chain, or by 2,4-dinitrophenol (2,4-DNP), an uncoupler of the oxidative phosphorylation. Sodium cyanide induced a transient increase of NADH fluorescence and 2,4-DNP decreased intracellular NADH fluorescence. Furthermore, the repercussions of cortical spreading depressions (CSD), a response of the brain to noxious stimuli, on cortical NADH fluorescence were determined. A single CSD decreased cortical NADH fluorescence for about 1 min, followed by a 5- to 10-min increase. The changes in NADH levels seem to correspond with the excitation and inhibition of neuronal metabolism, respectively. In summary, the measurement of NADH fluorescence using the laser technique allows the determination of changes in oxidative phosphorylation with high regional selectivity and time resolution.

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.

Trends in infection morbidity in a pediatric oncology ward, 1986-1995.

BACKGROUND AND PROCEDURE: We retrospectively studied the type, severity, frequency, and outcome of febrile infectious complications in 217 cancer patients receiving cytotoxic chemotherapy (603 episodes) over a 10-year period in a single pediatric institution. RESULTS: A total of 48.8% of the episodes occurred in severely leukopenic patients (WBC < 1.0 x 10(9)/l, absolute neutrophil count < 500 x 10(6)/l). In the second half of the study period febrile episodes occurred at increased frequency. The number of patients with gram-positive isolates in blood cultures increased over the years, most frequently coagulase-negative staphylococci were found. Remarkably, gram-negative bacteria increasingly resistant to the administered first-line antibiotic regimen emerged, necessitating modifications of the antimicrobial strategy every 3 years. Furthermore, Clostridium difficile-associated enterocolitis posed a clinical problem at increasing frequency since 1993. As expected, the speed of leukocyte recovery within 5 days from the onset of a febrile complication had an influence on the outcome of these episodes. CONCLUSIONS: Rapid recovery of the WBC was associated with an excellent prognosis whereas persisting neutropenia was found to be a negative factor associated with fatal outcomes. The fatality rate of all febrile episodes (2.3%) remained the same throughout the study period despite the availability and wider use of recombinant hematopoietic growth factors since 1991.

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.

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.

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.