Franz-Martin Fink

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.

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.

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.

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.

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.

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.

Outcome of children with primary resistant or relapsed non-Hodgkin lymphoma and mature B-cell leukemia after intensive first-line treatment: a population-based analysis of the Austrian Cooperative Study Group.

Children and adolescents with Non‐Hodgkin lymphoma (NHL) and mature B‐cell leukemia (B‐ALL) have an excellent prognosis with contemporary chemotherapy stratified according to the histologic subtype and clinical stage of disease. However, a small subset of patients does not respond to front‐line therapy or suffers from an early relapse.

Long-term outcome of initially homogenously treated and relapsed childhood acute lymphoblastic leukaemia in Austria - A population-based report of the Austrian Berlin-Frankfurt-Munster (BFM) Study Group

Relapsed acute lymphoblastic leukaemia (ALL) is the most common cause for a fatal outcome in paediatric oncology. Although initial ALL cure rates have improved up to 80%, the prognosis of recurrent ALL remains dismal with event‐free‐survival (EFS) rates about 35%. In order to analyse a population‐based cohort with uniform treatment of initial disease, we examined the outcome of children suffering from relapsed ALL in Austria for the past 20 years and the validity of the currently used prognostic factors (e.g. time to and site of relapse, immunophenotype). Furthermore, we compared survival rates after chemotherapy alone with those after allogeneic stem cell transplantation (SCT). All 896 patients who suffered from ALL in Austria between 1981 and 1999 were registered in a prospectively designed database and treated according to trials ALL‐Berlin‐Frankfurt‐Münster (BFM)‐Austria (A) 81, ALL‐A 84 and ALL‐BFM‐A 86, 90 and 95. Of these, 203 (23%) suffered from recurrent disease. One‐hundred‐and‐seventy‐two patients (85%) achieved second complete remission. The probability of 10‐year EFS for the total group was 34 ± 3%. Clinical prognostic markers that independently influenced survival were time to relapse, site of relapse and the immunophenotype. Additionally, a Cox regression model demonstrated that allogeneic SCT after first relapse was associated with a superior EFS compared with chemo/radiotherapy only (hazard ratio = 0·254; P = 0·0017).

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.