H-ficolin serum concentration and susceptibility to fever and neutropenia in paediatric cancer patients.

H-ficolin (Hakata antigen, ficolin-3) activates the lectin pathway of complement similar to mannose-binding lectin. However, its impact on susceptibility to infection is currently unknown. This study investigated whether the serum concentration of H-ficolin at diagnosis is associated with fever and neutropenia (FN) in paediatric cancer patients. H-ficolin was measured by time-resolved immunofluorometric assay in serum taken at cancer diagnosis from 94 children treated with chemotherapy. The association of FN episodes with H-ficolin serum concentration was analysed by multivariate Poisson regression. Median concentration of H-ficolin in serum was 26 mg/l (range 6-83). Seven (7%) children had low H-ficolin (< 14 mg/l). During a cumulative chemotherapy exposure time of 82 years, 177 FN episodes were recorded, 35 (20%) of them with bacteraemia. Children with low H-ficolin had a significantly increased risk to develop FN [relative risk (RR) 2.24; 95% confidence interval (CI) 1.38-3.65; P = 0.004], resulting in prolonged duration of hospitalization and of intravenous anti-microbial therapy. Bacteraemia occurred more frequently in children with low H-ficolin (RR 2.82; CI 1.02-7.76; P = 0.045). In conclusion, low concentration of H-ficolin was associated with an increased risk of FN, particularly FN with bacteraemia, in children treated with chemotherapy for cancer. Low H-ficolin thus represents a novel risk factor for chemotherapy-related infections.

Serum levels of mannose-binding lectin and the risk of fever in neutropenia pediatric cancer patients.

BACKGROUND: Fever in neutropenia (FN) is a frequent complication in pediatric oncology. Deficiency of mannose-binding lectin (MBL), an important component of innate immunity, is common due to genetic polymorphisms, but its impact on infections in oncologic patients is controversial. This study investigated whether MBL serum levels at cancer diagnosis are associated with the development of FN in pediatric cancer patients. PROCEDURE: Serum MBL was measured using ELISA. Frequency, duration, and cause of FN were assessed retrospectively. Association with MBL level was analyzed using uni- and multivariate Poisson regression taking into account both intensity and duration of chemotherapy. RESULTS: In 94 children, with a cumulative follow-up time of 81.7 years, 177 FN episodes were recorded. Patients with both very low MBL levels (<100 microg/L; risk ratio (RR), 1.93; 95% CI, 1.14-3.28; P = 0.014) and normal MBL levels (>or=1,000 microg/L; RR, P = 0.011) had significantly more frequent FN episodes than patients with low MBL levels (100-999 microg/L). Patients with very low MBL levels had significantly more episodes of FN with severe bacterial infection (bacteremia or pneumonia; RR, 4.49; 1.69 = 11.8; P = 0.003), while those with normal MBL levels had more FN episodes with no microbial etiology identified (RR, 1.85; 1.14 = 3.03; P = 0.014). CONCLUSIONS: Very low MBL levels are associated with more frequent FN episodes, mainly due to severe bacterial infections. The surprising finding that children with normal MBL levels had more frequent FN episodes than those with low MBL levels needs testing in prospective studies.

CDK2 catalytic activity and loss of nuclear tethering of retinoblastoma protein in childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. During cell cycle progression from the mid- to late G1 phase, mammalian cells traverse the restriction point, a transition from mitogen dependence to mitogen independence, regulated by retinoblastoma protein (Rb). Different cyclin-dependent kinases (CDKs) sequentially phosphorylate and inactivate Rb, which is associated by a change in Rb's nuclear affinity and activation of E2F transcription. Here, we show by in vitro kinase assays that ALL extracts contained CDK2 catalytic activity. When liberation of Rb from cell nuclei was tested by immune precipitation of differential cell extractions and Western blot analysis, little Rb was associated with the nuclear compartment. Together with the immunocytochemical analysis at a single cell level that Rb was phosphorylated at serine 612 and threonine 821, sites known to be phosphorylated by CDK2, the data indicated the presence of CDK2 catalytic activity and loss of Rb's nuclear affinity in ALL cells. We conclude that most ALL cells reside at or beyond the restriction point. This could explain the resistance of ALL cells to differentiation induction.

Cerebral sinovenous thrombosis during asparaginase treatment. Case 3.

A boy (age: 7 1/2 years) with acute lymphoblastic leukaemia developed thrombosis of the sinus sagitalis superior with secondary haemorrhagic infarction while on induction treatment with vincristine, prednisone, and asparaginase. Based on this report, the potential pathogenic mechanisms are discussed with respect to congenital prothrombotic defects as well as to the role of antileukaemic treatment. Current hypotheses on mechanisms for thromboembolism in children and proposed prophylactic strategies are briefly summarized.

Intellectual outcome in children and adolescents with acute lymphoblastic leukaemia treated with chemotherapy alone: age- and sex-related differences.

One of the most relevant concerns in long-term survivors of paediatric acute lymphoblastic leukaemia (ALL) is the development of neuropsychological sequelae. The majority of the published studies report on patients treated with chemotherapy and prophylactic central nervous system (CNS) irradiation, little is known about the outcome of patients treated with chemotherapy-only regimens. Using the standardised clinical and neuropsychological instruments of the SPOG Late Effects Study, the intellectual performance of 132 paediatric ALL patients treated with chemotherapy only was compared to that of 100 control patients surviving from diverse non-CNS solid tumours. As a group, ALL and solid tumour survivors showed normal and comparable intellectual performances (mean global IQ 104.6 in both groups). The percentage of patients in the borderline range (global IQ between 70 and 85) was comparable and not higher as expected (10% cases and 13% controls, expected 16%). Only 2 (2%) of the former ALL and 1 (1%) of the solid tumour patients were in the range of mental retardation (global IQ<70). Former known risk factors described in children treated with prophylactic CNS irradiation, like a younger age at diagnosis of ALL and female gender, remained valid in chemotherapy-only treated patients. The abandonment of prophylactic CNS irradiation and its replacement by a more intensive systemic and intrathecal chemotherapy led to a reduction, but not the disappearance of late neuropsychological sequelae.

Phosphorylation of the Retinoblastoma Protein in Childhood Acute Lymphoblastic Leukemia.

The cell cycle distribution of childhood acute lymphoblastic leukemia (ALL) cells shares many common features with that of normal CD34(+) progenitor cells. The majority of ALL and CD34(+) cells reside in G1 phase, although their cell cycle distribution within the G1 phase may be different. Traverse of the restriction point in late G1 phase is controlled by phosphorylation of the retinoblastoma protein (pRb). Different phosphoforms of pRb were recognized by their slower mobility on SDS-PAGE in ALL cells, while CD34(+) cells only contained the fast migrating p110Rb. Phosphorylated residues of pRb were detected only in ALL cells by immunoblotting with phosphospecific antibodies. Phosphorylation of pRb was due to cyclinD-Cdk4/6 protein kinase at Ser-608, Ser-780 and Ser-795. In contrast, phosphorylation at Ser-249/Thr-252, Thr-373, Ser-807/811 varied between ALL samples. No phosphorylation of pRb was detected at these residues in CD34(+) cells by immunoprecipitation. We concluded that the G1 phase distribution of ALL and CD34(+) was different, ALL cells progressed to mid G1 phase while unstimulated CD34(+) resided in early G1 phase.

In childhood acute lymphoblastic leukemia the hypophosphorylated retinoblastoma protein, p110RB, is diminished, as compared with normal CD34+ peripheral blood progenitor cells.

Acute lymphoblastic leukemia (ALL) of childhood arises from dysregulated clonal expansion of immature lymphoid precursor cells that fail to differentiate into functional lymphocytes. The cell-cycling status of ALL cells shares many common features with that of normal CD34+ hematopoietic progenitor cells, such as low number of resting G0 and cycling S phase cells even though the growth fraction is high. Thus, ALL cells should be in a long G1 phase. Phosphorylation of the retinoblastoma protein is a crucial step in cell-cycle progression from G0/early G1 to late G1/S phase. We therefore analyzed the G1 distribution of these two immature cell populations by immunostaining and Western blot. Bone marrow samples from children with ALL at diagnosis as well as purified CD34+ cells, before and after in vitro stimulation with cytokines, were investigated for the expression of hypophosphorylated p110RB (early G1 phase), total retinoblastoma protein, statin (G0 phase), bromo-deoxyuridine (S phase), proliferating cell nuclear antigen, and p120 (cycling cells). Compared with unstimulated CD34+ cells (95.8 +/- 1.2%) the component of ALL cells containing hypophosphorylated p110RB (16.3 +/- 13.2%) was significantly reduced (p = 0.00018), whereas only a minor difference could be detected for the proportion of cycling cells (p = 0.03), and no difference in G0 and S phase cells (p > 0.05). Our results indicate that, as opposed to unstimulated CD34+ cells, the majority of ALL cells are beyond the restriction point and therefore irreversible committed to DNA replication and mitosis.

Liver iron and fibrosis during long-term treatment with deferiprone in Swiss thalassaemic patients.

Serum ferritin levels, hepatic histology and iron concentration were studied in a 'veteran' group of seven Swiss beta-thalassaemic patients after 93-99 months of treatment with the oral iron chelator deferiprone (L1), and another four patients who had received 54-82 months of L1 therapy. Despite continuous compliance, unexplained resurgence of serum ferritin levels occurred in 4/7 patients of the 'veteran' group after 4-5 years on L1. In three of these a concomitant increase of liver iron was also observed. Hepatic histology revealed significantly higher degrees of fibrosis in 6/11 hepatitis C (HC)-positive patients (fibrosis scores 1-5, mean 3.0) than in the HC-negative group (fibrosis score 0-2, mean 0.8). Two HC-negative patients had no detectable fibrosis after 98 and 93 months on deferiprone. Therefore the hepatic pathology in these patients cannot definitely be attributed as a side-effect of deferiprone. Chronic active hepatitis C and the accumulation of iron are the major causative factors to be considered.

Differential phosphorylation of lamin B2 in normal and leukemic cells.

Lamins constitute the nuclear lamina, which underlie the inner membrane of the cell nucleus. Phosphorylation of lamins is a key factor in the regulation of nuclear structure during the cell cycle and of gene transcription. Since an uncontrolled cell cycle and altered gene transcription are major characteristics of neoplasms, we looked for differences in lamin B2 phosphorylation between PBMC, ALL and AML cells. Using different lamin B2-specific antibodies, we detected two different lamin B2 species termed lamin B2 and B2A. Although phosphorylation of lamin B2 in leukemic cells was reminiscent of resting cells, the majority of ALL and AML samples showed significantly higher and more altered lamin B2A phosphorylation compared to PBMC. It remains to be elucidated which mechanism leads to these alterations and whether it could explain the extended G1-phase frequently observed in ALL cells.

High incidence of significant bone loss in patients with severe congenital neutropenia (Kostmann's syndrome).

OBJECTIVE: Clinical observation of bone pain, unusual fractures in two patients, and diffuse osteopenia/osteoporosis led us to assess bone mineral content and density in 30 patients with severe congenital neutropenia who were treated with recombinant-methionyl-human granulocyte colony-stimulating factor (r-metHuG-CSF). STUDY DESIGN: We reviewed roentgenograms in 29 of these 30 patients to evaluate bone loss before and during treatment. In addition, in 17 of the 30 patients, bone mineral status could be assessed by both quantitative computed tomography (Q-CT; n = 16) and dual energy x-ray absorptiometry (DXA; n = 1). In one patient, Q-CT was not possible because of severe vertebral fractures. RESULTS: Of the 30 patients investigated, 15 had evidence of osteopenia/osteoporosis observed on spine radiographs (n = 5), on Q-CT/DXA (n = 1/n = 1), or on radiographs and Q-CT (n = 8). In 13 of the 30 patients, only a lateral radiograph of the lumbar spine was available, 5 of 13 showing either increased kyphosis and wedging of the vertebrae or compression fractures of the vertebral bodies, indicating severe established osteoporosis. In eight patients, the findings of the spinal radiographs were normal. In nine patients, spinal radiographs were taken before r-metHuG-CSF treatment. Osteoporotic vertebral deformation (n = 3) or reduced bone mass (n = 3) was seen in six of these nine patients. The levels of serum biochemical markers of bone metabolism were all within normal ranges except for mild elevation of the serum alkaline phosphatase level. The degree of spinal bone mineral loss did not correlate with dose and duration of r-metHuG-CSF treatment or with the age or sex of the patients. CONCLUSIONS: These data indicate a high incidence of bone mineral loss in children with severe congenital neutropenia. The underlying pathogenesis of bone demineralization is not clear. It is more likely that the bone loss was caused by the pathophysiologic features of the underlying disease, but it is possible that r-metHuG-CSF accelerates bone mineral loss.

Cell birth and death in childhood acute lymphoblastic leukaemia: how fast does the neoplastic cell clone expand?

In 23 children with untreated precursor B-cell acute lymphoblastic leukaemia (ALL), the daily growth rate of the malignant cell clone was calculated. Cell birth expanded the leukaemic cell clone an average 10-11% per day, programmed cell death or apoptosis reduced the leukaemic cell mass by some 4% per day. From these two variables a net increase in the size of the leukaemic cell population of 6.9+/-7.3% (range -1.2-27.3%) per day could be calculated. The daily growth rate correlated negatively with the logarithm of the duration of clinical symptoms before the diagnosis of ALL was established (r=-0.680; P=0.0004). A long history, especially in children with undefined bone pain and arthralgias, was associated with a very slow expansion of the neoplastic cell clone.

[Doping in animal rights law: the responsibility of the veterinary surgeon]. / Doping im Tierschutzrecht: die Verantwortlichkeit des Tierarztes.

Unlike the situation in humans, it is illegal to administer doping drugs to animals. The Doping Commission of the European Council has defined the term doping. According to this definition, the therapeutic use of drugs is not doping. The treatment of pain is also therapeutic. However, governing bodies of sport can apply stricter regulations (doping lists). It is possible for veterinarians to infringe paragraph 3 (11) of the German animal welfare act if they act recklessly or without due care, and a prosecution may result. It is therefore obligatory for the veterinarian to advise of the possible consequences of administering a drug which has a doping effect, or is included on a doping list.

Acute lymphoblastic leukaemia in childhood: cell proliferation without rest.

The percentage of non-cycling blast cells in children with untreated acute lymphoblastic leukaemia (ALL) was investigated by staining smears for statin, a nuclear protein specifically present in non-growing resting cells. Results were compared with purified normal CD34-positive progenitors. A low fraction of ALL and CD34-positive cells expressed statin (2.9 +/- 3.8% and 2.8 +/- 3.1%, respectively), the growth fraction assessed by staining for the nucleolar antigen p120 was 94% in both ALL and CD34-positive cell samples. From this analysis it can be concluded that the compartment of non-replicating cells in ALL as well as in normal CD34-positive precursor cells collected from peripheral blood is very small and that most cells are cycling.

Tunneled, double lumen Broviac catheters are useful, efficient and safe in children undergoing peripheral blood progenitor cell harvesting and transplantation.

We prospectively evaluated performance, efficiency, safety and compliance of large-bore central venous catheters (Cook TPN; Cook (Switzerland) AG) introduced via saphenous veins in 32 children and infants elected to undergo peripheral blood progenitor cell (PBPC) harvesting and transplantation (PBPCT). With these catheters a flow rate (25-65 ml/min) adequate for leukapheresis was achieved in all patients. There were no important catheter-related complications during harvest. The total duration of catheter placement was 4569 days (median, 139 days; range, 8-268 days). During this period which included conditioning and PBPCT, we observed five mechanical complications and 12 septic episodes not necessarily catheter-related (0.11 and 0.26 events per 100 catheter days, respectively). All infections resolved after systemic antibiotic treatment. There was no exit or tunnel area infection, and no catheter had to be removed due to infection. Two catheters were replaced because of displacement. Tunneled double lumen Broviac catheters introduced via saphenous veins were not only efficient and safe, they were also well accepted by children and young adults undergoing PBPC harvesting and transplantation.

Focal nodular hyperplasia of the liver in children: review of follow-up and outcome.

The authors used a conservative approach to treat focal nodular hyperplasia of the liver (FNH) and were interested in the long-term results of different therapeutic approaches in order to establish the most appropriate treatment. A review of the literature was conducted, and 31 pediatric case reports were identified in which follow-up and outcome data were noted. Tumor resection was performed in 18 cases (58%), operative biopsy alone in nine (29%), vascular ligation in two, and embolization of hepatic arteries in two. The outcome appears to be good for both groups of patients, ie, those with observation alone and those with resection. More information is to be collected regarding the new procedures (embolization and ligation). This is a retrospective analysis, and data from future international prospective studies are needed.

Incidence of childhood acute lymphoblastic leukemia (ALL) and population-based treatment results in Switzerland: experiences with 507 study and 149 nonstudy patients.

Of 656 patients with ALL (all types) diagnosed in Switzerland during 4 consecutive 4-year periods (1976-1979, 1980-1983, 1984-1987, 1988-1991), 507 were officially registered on protocols ("study" patients) while 149 were not ("nonstudy" patients). The mean incidence of 3.8/100,000 children < 15 years/year is higher than reported for other Western countries. Evidence is presented suggesting that the 656 patients represent only approximately 90% of all children with ALL residing in Switzerland, indicating that the true incidence of ALL might even be higher. The fraction of "nonstudy" patients fell from 40% (1976-1979) to 15% (1984-1987). The rate of survival at 4 years of all patients with ALL ("study" and "nonstudy") increased by 17% during the three consecutive periods 1976-1979, 1980-1983, and 1984-1987. As expected, a higher increase (20%) was observed in "study" patients and a statistically nonsignificant lower one (10%) in "nonstudy" patients.

Childhood NHL in Switzerland: incidence and survival of 120 study and 42 non-study patients.

Based on the Swiss Pediatric Oncology Group (SPOG) cancer registry data during 1981-1991, a high average incidence of 8 new NHL per million children younger than 15 years per year was found. Of 162 children with NHL registered in 1976-1991, 120 were study patients, i.e., officially registered and treated according to SPOG or Pediatric Oncology Group (POG) protocols, while 42 were non-study patients, i.e., patients not officially enrolled on protocols. Overall, 91 of 120 (76%) study patients remained alive. Seventy-nine study patients were treated according to older SPOG protocols, and 53 (67%) of these survived, while 38 of 41 (93%) study patients treated according to newer POG protocols remained alive (P = 0.0068). Only 22 (52%) of the 42 non-study patients survived (P = 0.0001). There was no improvement if the survival of non-study patients before and since 1986 was compared. Population-based treatment results in Switzerland were similar to those in the United Kingdom. They provided an important base for the development of future treatment strategies.

The number of circulating CD34+ blood cells predicts the colony-forming capacity of leukapheresis products in children.

In children, only a few guidelines are available for optimizing peripheral blood progenitor cell (PBPC) harvesting. We analyzed by means of flow cytometry and clonogenic assays 60 harvest products obtained from 20 children by standardized leukapheresis after treatment with chemotherapy and CSF. In addition, 27 fresh blood samples obtained prospectively during the mobilization phase were studied. CFU-GM/kg significantly correlated with MNC/kg, CD34+ cells/kg and CD34+33- cells/kg in apheresis products (P < 0.001). In fresh blood samples, CFU-GM/ml significantly correlated with MNC/ml, CD34+ cells/ml and CD34+33- cells/ml (P < 0.001). The numbers of CD34+ cells/ml, CD34+33- cells/ml and MNC/ml in 19 blood samples taken prior to leukapheresis were compared with CFU-GM/kg harvested and thawed after cryopreservation applying multiple regression analysis with stepwise variable selection. The number of circulating CD34+ cells/ml prior to leukapheresis highly correlated with and was predictive for the number of collected CFU-GM/kg (P < 0.001). In addition, a significant correlation (P < 0.05) between the number of progenitor cells/kg reinfused and the time to myeloid and platelet recovery was found in children undergoing high-dose therapy. Our data indicate that a single leukapheresis will be sufficient to obtain a minimum number of 5 x 10(4) CFU-GM/kg if the pre-harvest number of circulating CD34+ cells is > or = 10(5)/ml. Thus, our results will help to optimize PBPC transplantation in children.