Prospective study of thromboembolism in 1038 children with acute lymphoblastic leukemia: a Nordic Society of Pediatric Hematology and Oncology (NOPHO) study.

UNLABELLED: ESSENTIALS: Children with acute lymphoblastic leukemia (ALL) are at risk of thromboembolism (TE). This is a prospective evaluation of the incidence, risk factors and outcomes of TE in 1038 children with ALL. TE occurred in 6.1% of children, with the highest incidence (20.5%) among those aged 15-17 years. A TE-associated case fatality of 6.4% indicates that TE is a severe complication of ALL treatment. BACKGROUND: Thromboembolism (TE) is a major toxicity in children with acute lymphoblastic leukemia (ALL) and may have a negative impact on ALL treatment. OBJECTIVES: To examine the cumulative incidence, outcomes and risk factors associated with TE in children with leukemia. PATIENTS/METHODS: We prospectively evaluated TE in 1038 Nordic children and adolescents (≥ 1 and < 18 years) diagnosed with ALL during 2008-2013 and treated according to the NOPHO (Nordic Society of Pediatric Hematology and Oncology)-ALL 2008 protocol. The cohort was followed until December 2014. Cox proportional regression was used to compute hazard ratios (HRs). RESULTS: TE events (n = 63) occurred most frequently in conjunction with asparaginase (ASP) administration (52/63). The cumulative incidence of TE was 6.1% (95% confidence interval [CI], 4.8-7.7). Being aged 15-17 years was associated with an increased risk of TE (adjusted HR of 4.0; 95% CI, 2.1-7.7). We found a TE-associated 30-day case fatality of 6.4% (95% CI, 1.8-15.5) and TE-related truncation of ASP therapy in 36.2% (21/58). Major hemorrhage occurred in 3.5% (2/58) of anticoagulated patients. Minor hemorrhage was reported in two out of 58 patients. No major bleeds occurred in children who received low-molecular-weight heparin. CONCLUSIONS: Methods to identify children and adolescents who will benefit from thromboprophylaxis during ALL treatment are called for. The truncation of ASP should be avoided. The long-term survival outcomes for ALL patients with TE require close monitoring in the future.

Paediatric arterial ischaemic stroke and cerebral sinovenous thrombosis in Denmark 1994-2006: a nationwide population-based study.

AIM: To assess the incidence rates (IR), clinical characteristics, risk factors, treatment and outcomes of paediatric arterial ischaemic stroke (AIS) and cerebral sinovenous thrombosis (CSVT). METHODS: Using population-based, nationwide medical registries, we identified all patients aged 0-18 years at the time of hospitalization with first-ever AIS and/or CSVT in Denmark between 1994 and 2006. Medical records were retrieved and reviewed. RESULTS: We identified 211 patients with AIS and 40 patients with CSVT corresponding to IRs of 1.33 (95% CI 1.16-1.52) and 0.25 (95% CI 0.19-0.34) per 100,000 person-years, respectively. The IRs peaked in infancy (<1 year) for both AIS and CSVT with an additional peak among adolescents (15-18 years) for CSVT. The IR of AIS increased 3.9% per year (p=0.036), whereas no changes were found for CSVT. In total, 48.2% of the patients received antithrombotic treatment; no major complications were observed. All-cause and thrombosis-related 30-day case fatality ratios were 3.6% and 2.4%, respectively; neurological sequelae were found in 56.2% of patients. CONCLUSION: The IR of AIS was highest in infants and had increased with 3.9% annually during the observation period. The IR of CSVT had an additional peak in adolescence and remained unchanged over time.

Immune thrombocytopenic purpura in childhood in Norway: a prospective, population-based registration.

A prospective, population-based registration of children with immune thrombocytopenic purpura (ITP) was performed in Norway in 1996 and 1997. Ninety-two cases were identified, indicating an incidence of 5.3 per 100,000 children under 15 years. The sex ratio (female/male) was 1.2/1. Fifty-six percent presented with cutaneous signs only. The lowest platelet count was < 20 x 10(9)/L in 91%. In spite of mild bleeding symptoms, medical treatment was given in 68%, in most cases (57/63) with intravenous immunoglobulin. A total of 41/44 patients with platelet counts of < or = 5 x 10(9)/L were treated, regardless of whether they had mucous bleedings or not. Eighteen percent had platelet counts < 150 x 10(9)/L at 6 months, and 9% at 12 months following diagnosis. One patient with therapy-resistant chronic ITP died 16 months after diagnosis from an anesthesia complication related to profound epistaxis. This study shows a relatively high incidence. As in other studies, there was a tendency to treat platelet counts rather than bleeding symptoms.

Prolonged intrathecal chemotherapy replacing cranial irradiation in high-risk acute lymphatic leukaemia: long-term follow up with cerebral computed tomography scans and endocrinological studies.

UNLABELLED: Cranial irradiation in children with acute lymphatic leukaemia (ALL) decreases the risk of CNS relapse but is associated with serious long-term side-effects. We present the long-term outcome of 21 children with high-risk ALL who received prolonged intrathecal chemotherapy instead of the recommended cranial irradiation. Intrathecal triple therapy (methotrexate, hydrocortisone, and cytarabine) was administered every 2nd month throughout the maintenance phase. The average number of courses of intrathecal methotrexate was 8.7 and of triple 9.0. The 5-year event-free survival was 79%. No CNS relapses occurred. CT scan was performed at diagnosis, at cessation of therapy, and 3 years thereafter. No density abnormalities, pathological contrast enhancement, ventricular dilatation, or calcifications were found. One child showed cortical atrophy both at diagnosis and at cessation of therapy. There was a slight decrease in height SDS with time but no change in weight SDS. Delayed bone age was found in 5 children. No abnormalities of growth hormone, thyroid, adrenal, or gonadal function were observed. CONCLUSION: The study indicates that extended intrathecal chemotherapy in children with high-risk ALL may provide an effective protection from CNS relapses and is associated with a low risk of long-term side-effects.

The impact of 123I-meta-iodobenzylguanidine scintigraphy on diagnostics and follow-up of neuroblastoma.

The present retrospective study includes all children suspected for having neuroblastoma, admitted to Odense University Hospital in September 1984 through December 1993. Thirty-six children at the age range of 1 month to 14 years and 10 month were investigated with 123I-metaiodobenzylguanidine (MIBG). Nineteen children had histologically verified neuroblastoma. Several MIBG scintigraphic examinations were made in all but one of these 19 children. Positive MIBG scintigraphy strengthened the diagnosis and indicated the volume and location of the tumour at diagnosis and during the treatment period. In a few patients only there was some disagreement between results obtained with MIBG scintigraphy and standard investigations as CT-scanning or ultrasonography. MIBG scintigraphy in all cases turned out to be the most sensitive modality.

[Late endocrine and growth sequelae after cancer treatment in children]. / Endokrine og vaekstmaessige senfølger efter cancerbehandling af børn.

Growth and endocrinological disturbances are possible late side-effects of cancer treatment in childhood. These side-effects can be treated, thus their discovery is important. The side-effects particularly appear in the years following treatment with irradiation and/or alkylating chemotherapy. After irradiation of the brain or the neck the function of the thyroid and the parathyroid glands should be tested every third month the first year, and later on annually. Two years after the end of treatment, the patient should be examined for growth hormone deficiency. This examination should be carried out annually. One should be alert to symptoms of pubertas praecox the years prior to puberty. At the age when puberty is expected and thereafter one should look for signs of secondary hypogonadism. Primary hypogonadism may follow radiotherapy below the diaphragm and/or treatment with alkylating chemotherapeutics; further, reduced fertility in men and early menopause in women may follow these treatments. The bone structure of the face and the teeth may be damaged by radiation and chemotherapy, so therefore yearly examination by a dentist with specialty in this subject is recommended. Surgery in order to improve function may be a possibility.

Purification of human blood basophils by negative selection using immunomagnetic beads.

A simple and rapid method for isolating highly purified and functionally intact basophils from normal human blood is described. Blood from healthy volunteers was centrifuged through a two layer Percoll density gradient. The majority of the basophils were recovered between Percoll layers with densities of 1.070 and 1.080, constituting 45.6% of total leukocytes. Lymphocytes, monocytes and neutrophils were extracted from this fraction using a panel of mabs in a direct or indirect selection procedure using immunomagnetic beads (Dynabeads) coated with sheep anti-mouse IgG. These negative immunoselection procedures produced a high yield of basophils with a mean purity of 97.8% (range 93.0-99.5%) and 97.0% (range 96.2-99.0%) using the direct and indirect method, respectively. The cells isolated by this method are viable, release histamine to various stimuli in a normal manner, and appear morphologically normal in transmission electron microscopy.

Characterization of a new malignant human T-cell line (PFI-285) sensitive to ascorbic acid.

A new malignant human T-cell line-labelled PFI-285-has been isolated from a boy with malignant lymphoma. Morphologically, the cells had characteristics of malignant lymphoid cells. The cells presented surface antigens as early cortical lymphocytes and proliferated non-adherently as single cells, independent of T-cell growth factor (IL-2), in liquid culture. The cells had undetectable levels of receptors for IL-2, were not clonogenic in soft agar, but did form tumors in nude mice. Their establishment and continuous growth in vitro was dependent on the number of cells inoculated and on the growth medium used. Cytogenetic alteration, HTLV-1 or reverse transcriptase activity were not detected. The production of known T-cell derived lymphokines such as IL-2, B-cell growth factor(s), alpha-interferon or granulocyte/macrophage colony stimulating or inhibiting factor(s) was not detected. The cells had 5-8% natural killer (NK)-cell activity against NK-cell sensitive target cells (K562) and were not sensitive for NK cells. A most unusual characteristic was the pronounced sensitivity of the cells to ascorbic acid. Concentrations down to 50 mumol/l killed the cells within hours.

Human granulopoiesis in vitro--medium-dependent growth regulation by a granulocyte-derived inhibitor.

A number of reports have indicated that mature blood granulocytes produce regulators that inhibit proliferation of progenitor cells in the bone marrow. However, this concept of negative feedback of granulopoiesis is still controversial. To examine whether conflicting results may depend upon the experimental set-up, we have compared colony formation by human bone marrow cells in different growth media. Unmodified McCoy's medium, which in feeder layer cultures supports the formation of large numbers of colonies, was a poor growth medium in cultures supplied with crude or recombinant colony stimulating factor (CSF). The colony formation improved when the medium was supplemented with defined additives. In CMRL 1066 cultures, granulocyte extract (GRE) consistently caused a strong inhibition of colony formation. In contrast, with unmodified McCoy's medium, granulocyte extract enhanced colony formation in a dose-dependent manner. The enhancing effect of granulocyte extract coincided with low colony numbers in the control cultures. The stimulatory effect of granulocyte extract in McCoy's medium, switched to strong inhibition when thymidine, a component of CMRL 1066 medium, was added. The inhibitory and stimulatory activities were found in the same molecular weight fractions (30-60 kD) after gel filtration. Both modulators in granulocyte extract appeared to be independent of monocytes and T lymphocytes in the bone marrow, as shown by removal of these cells with magnetic microspheres coated with specific monoclonal antibodies. The present work shows that regulation of cell proliferation in vitro depends strongly on culture conditions, such as choice of medium. It appears that thymidine acts as co-factor for the inhibitor in granulocyte extract.

Thymidine-dependent effect of granulocyte-derived inhibitor on granulocyte-macrophage progenitor cells (GM-CFC).

We have compared the effect of granulocyte extract (GRE) on proliferation of hemopoietic cells from various sources, using two different cultures media, CMRL 1066 and McCoy 5A. GRE caused a strong (80-90%) inhibition of granulocyte-macrophage colony forming cells (GM-CFC) in cultures with medium CMRL 1066. GM-CFC in human blood and human and mouse bone marrow were equally sensitive to the inhibitor. The inhibitor had a maximal effect in the concentration range corresponding to GRE from 2 x 10(5) to 2 x 10(6) cells per 1 ml culture dish. At higher GRE concentration the inhibition was reduced. GM-CFC from human blood and mouse marrow were suppressed in cultures with McCoy's medium as well, but to a lesser extent than in CMRL 1066 cultures. On the other hand, in cultures with human bone marrow cells (BMC) and McCoy's medium, GRE had no inhibitory effect. CMRL 1066 medium contains a number of components not present in McCoy's medium. In a systematic study where these substances were added one by one to McCoy's medium we found that inhibition by GRE depended upon the presence of thymidine. At a thymidine concentration of 3 x 10(-5) mol/l GRE strongly suppressed GM-CFC in human blood and bone marrow. This thymidine concentration itself had no effect. Other nucleosides or components of the CMRL 1066 did not potentiate the suppressive effect of GRE.

Colony inhibiting factor in mature granulocytes from normal individuals and patients with chronic myeloid leukemia.

Inhibitory activity in extract from human blood granulocytes was tested on granulocyte-macrophage colony formation in vitro. The inhibition depended on the type of serum used. With mouse BMC and FCS in the cultures, extract corresponding to 2.5 X 10(4) granulocytes/ml reduced the colony number by 35%, and extract from 2 X 10(5) cells caused maximal inhibition (80-90%). With HS and mouse BMC the colony number was reduced by only 11-12%, but stronger inhibition (55%) was observed when the serum concentration was reduced. With both types of sera the total cell number per culture plate was reduced relatively more than the colony number. Human GM-CFC were as sensitive as mouse GM-CFC, and extract from CML granulocytes inhibited less (p less than 0.01) than extract from normal cells. Biochemical studies indicated that the inhibitor is a protein with a molecular weight of 30-60,000. Lactoferrin, a putative inhibitor of CSF production, did not inhibit spontaneous or CSF-induced colony formation in these studies.

Endogenous inhibitors of human granulocyte/macrophage colony forming cells in vitro are inactivated by free radical scavengers.

Granulocyte/macrophage colony forming cells (GM-CFC) require colony stimulating factors (CSF) for growth in vitro. Crude preparations of CSF also contain inhibitors. To investigate whether this inhibition involves free radicals, the following scavenging agents were tested on human GM-CFC grown in agar: cysteine, reduced glutathione, dithiothreitol, sodium selenite, alpha-tocopherol, superoxide dismutase, catalase and ascorbic acid. All except ascorbic acid and catalase had enhancing and stabilising effects on colony formation. The colonies also contained more cells but no changes in cell composition were detected. Cysteine, the most extensively tested substance, was active at concentrations present in body fluids and enhanced colony formation without an exogenous source of CSF. Our results are consistent with an imbalance between the production and removal of free radicals generated in the human GM-CFC assay in vitro. This hampers investigation of regulators of granulopoiesis and results in underestimation of GM-CFC, but can be alleviated by the addition of free radical scavengers.

Vitamin C and thiol reagents promote the in vitro growth of murine granulocyte/macrophage progenitor cells by neutralizing endogenous inhibitor(s).

Growth of murine hemopoietic cells in culture requires the presence of a stimulator of stem cell proliferation, "colony stimulating factor" (CSF). A widely used source of CSF is lung conditioned medium (LCM). We have earlier shown that the great variability of CSF activities in different batches of LCM is due to varying amounts of inhibitor(s). The present study expands the observation that the addition of ascorbic acid to the murine bone marrow soft agar assay system removes the inhibitory activity. The vitamin probably acts as an antioxidant or free radical scavenger, since addition of reduced (but not oxidized) glutathione, cysteine, dithiothreitol or 2-mercaptoethanol to the cultures also inactivates the endogeneous inhibitor. Cysteine and glutathione gave the highest colony numbers, were active at concentrations present in body fluids and did not inhibit colony growth even at concentrations ten times higher than optimum. No synergistic effects could be observed between the different antioxidants. At optimum concentration (usually 0.45 mmol/l) the otherwise bell-shaped dose-response curve for conditioned medium changed to a sigmoid curve. Antioxidants had no growth promoting effect in the absence of CSF. The presence of cysteine or vitamin C revealed CSF-like activity in conditioned media of tissues not considered to be potent producers of such factors. It has been reported that individual batches of foetal calf serum contain different levels of reduced glutathione, and we suggest that one of the batch variable growth regulators in foetal calf serum may be reduced glutathione. The results indicate a possible physiological role of antioxidants in granulopoiesis and suggest that cysteine or reduced glutathione should be freshly added to culture systems assaying CSF and/or granulocyte macrophage progenitor cells.

Inhibitory activity in mouse lung-conditioned medium studied in the agar assay for bone marrow colony-forming cells: removal by vitamin C.

The growth of granulocyte/macrophage colony-forming cells (CFC) from bone marrow, with mouse lung-conditioned medium (LCM) as source of colony-stimulating factor, was tested in five strains of mice. Maximum stimulation of growth was usually obtained at a LCM dilution of 1:5 (final concentration, 1.8% vol/vol). Higher concentrations nearly always caused inhibition. The inhibitory activity was resistant to heating (56 degrees C, 30 min) and was not removed by dialysis, but could be extracted with chloroform, suggesting a lipophilic inhibitor of high molecular weight. On gel filtration (Sephadex G50) the stimulatory activity eluted in the first fraction, whereas the inhibitory activity could not be recovered. Ascorbic acid reduced or prevented the inhibition (optimum concentration, 0.45 mmol/l), and provides a convenient means of stabilizing the CFC agar assay system. In physiological conditions the vitamin might influence the responsiveness of CFC to growth-regulating factors.