Lidija Kitanovski

First prospective report on immune tolerance in poor risk haemophilia A inhibitor patients with a single factor VIII/von Willebrand factor concentrate in an observational immune tolerance induction study

Development of neutralizing inhibitors against factor VIII (FVIII) is a major complication of haemophilia A treatment.

Multifocal hepatoblastoma in a 6-month-old girl with trisomy 18: a case report

IntroductionEdwards syndrome (trisomy 18) is a rare entity with a reported incidence of 1/3000 to 1/7000 births. Less than 10% of patients survive beyond the first year of life, which may influence the fact that malignant tumors are rarely reported in association with this syndrome.Case presentationThe authors report a rare case of a 6-month-old girl with trisomy 18 and multifocal hepatoblastoma. The course of the disease, autopsy results and review of the literature are presented.ConclusionOur case represents the seventh published case of hepatoblastoma in a patient with trisomy 18. All of the seven published cases were women, possibly due to the high preponderance of females among the children with Edwards syndrome and longer survival of females with trisomy 18 compared to males. Since both trisomy 18 and hepatoblastoma are rare conditions, the probability that a child with trisomy 18 will independently develop a hepatoblastoma is very low. Therefore, we believe that the existence of these cases in children with trisomy 18 indicates a significant association. It can be assumed that trisomy 18 potentiates the development of hepatoblastoma. Careful clinical and post-mortem studies are needed to recognize the real frequency of hepatoblastoma in children with trisomy 18, who might die from different causes with unrecognizable hepatoblastoma.

No evidence of association of methylenetetrahydrofolate reductase polymorphism with occurrence of second neoplasms after treatment of childhood leukemia.

Methylenetetrahydrofolate reductase (MTHFR) polymorphisms have been associated not only with the risk for acute lymphoblastic leukemia (ALL) in adults and children, but also with increased methotrexate toxicity. The present study aimed to investigate whether MTHFR polymorphisms modify the risk for development of secondary malignancies in children treated for ALL with protocols that included high-dose methotrexate. MTHFR genotypes were determined in DNA samples isolated from archived bone marrow smears of 15 patients with a second malignancy and a matched control group of 30 patients who did not developed a second malignancy after the treatment for ALL. The frequencies of MTHFR C677T and A1298C genotypes in all patients were: C677T: CC 40%, CT 46.7% and TT 13.3% and A1298C: AA 46.7%, AC 44.4% and CC 8.9%. The relative risk for second malignancy was not significantly increased in ALL patients having at least one polymorphic C667T [odds ratio (OR) 1.51; 95% confidence interval (CI) 0.43-5.31] or one polymorphic A1298C allele (OR 1; 95% CI 0.29?-?3.46). Our study suggests that MTHFR polymorphisms are not associated with increased risk of second cancer in children treated with high-dose methotrexate.

Bifidobacterium breve Sepsis in Child with High-Risk Acute Lymphoblastic Leukemia.

To the Editor: Patients with cancer often consume probiotics as part of their diet, although therapeutic use of probiotics is not recommended because of their potential invasiveness. In a recent review, 5 cases of probiotic treatment–related bacteremia were identified in oncology patients, although no cases of invasive Bifidobacterium spp. infection were included (1). We describe a case of B. breve sepsis in a child with Philadelphia chromosome–positive acute B-cell lymphoblastic leukemia. The patient was a previously healthy 2-year-old boy who had no history of immunodeficiency and whose leukocyte counts had been within reference ranges during check-up visits before his diagnosis. After leukemia was diagnosed, chemotherapy was started (prednisone, vincristine, doxorubicin, and L-asparaginase). During the second week of treatment, the boy experienced abdominal discomfort and constipation. Two weeks later, his condition worsened; he refused food, his abdomen was distended, and he had colicky pain. Thickened intestinal wall and fecal masses were seen ultrasonographically. Twelve hours later he became hypotensive. Laboratory test results showed severe neutropenia and increased inflammatory markers (Figure). Two aerobic and anaerobic blood culture samples were collected from a central venous line (implantable venous access system) in a 30-minute span, and treatment with piperacillin/tazobactam, vancomycin, and gentamicin was empirically initiated according to local recommendations for pediatric febrile neutropenia with shock. Both anaerobic blood cultures were positive. Gram-positive, irregular rods with bifid and branching forms without spores grew anaerobically on blood agar with hemin and vitamin K after 48 hours of incubation and were identified as B. breve by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Bruker Daltonics, Billerica, MA, USA). The bacteria were susceptible to penicillin (MIC 0.250 μg/mL), ampicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam (MIC 0.125 μg/mL), imipenem, and clindamycin but not metronidazole. Gentamicin and vancomycin were discontinued, and piperacillin/tazobactam was replaced by penicillin (Figure). The patient stayed afebrile, and his neutropenia resolved. A blood culture taken on the eighth day of antimicrobial drug treatment was negative, and the central venous line was not replaced at that time. Bowel movement normalized and was maintained. We reviewed the ingredients of the food that the child received and documented the presence of Lactobacillus spp. and B. longum but not B. breve. Figure Schematic presentation of leukocyte count, C-reactive protein, and procalcitonin serum levels in clinical course of Bifidobacterium breve sepsis. Arrows indicate the name and duration of each antimicrobial drug treatment. Some probiotics are part of the normal intestinal microbiota and rarely cause invasive infections (2). Although Bifidobacterium spp. is infrequently associated with infections (<5% of anaerobic isolates), it occasionally causes serious illness. On the rare occasions when it is isolated from patients with bloodstream infections, it is usually isolated along with other causative agents. The number of reported deaths associated with anaerobic nonsporulating gram-positive rods is low (3). In this patient, abdominal symptoms coincided with 2 blood cultures that yielded B. breve. We assume bacteria translocated through the distended colonic wall during chemotherapy-induced neutropenia and believe that the blood culture isolate was not a contaminant because it was isolated from 2 samples taken in a 30-min span. It is the practice at our institution not to take peripheral blood cultures simultaneously because doing so does not increase diagnostic accuracy. We found 1 description in the literature of B. breve septicemia in a neonate with omphalocele who had received probiotic therapy (4). In a review of Bifidobacterium spp. isolates during 2000–2007 in 2 US hospitals, B. breve was isolated from blood culture from 3 adult patients (5). Two of these infections were associated with ileal resection or peritonitis and 1 with decubitus ulcers. No data on antimicrobial drug treatment were available. Bifidobacterium spp. sepsis was reported in an infant of extremely low birthweight 10 days after probiotic supplementation who recovered after antimicrobial drug therapy, although stenosis of the colon developed 6 weeks later (6). Blood culture grew B. longum and B. infantis, which were probiotic strains. Apart from 1 case of sepsis caused by B. longum associated with acupuncture in a 19-year-old healthy patient (7), we did not find other reports of invasive Bifidobacterium spp. infections. Because neutropenic episodes, even with bowel involvement, are common during treatment for cancer (8), no reason to promote therapeutic use of probiotics has been proven. Probiotics can cause substantial bacterial overgrowth when stimulating factors are present. In our opinion, avoiding fecal impaction is crucial for preventing colonic bacterial overgrowth and minimizes the chance that bacteria will translocate and cause invasive infection. Nutritional recommendations for a neutropenic diet for children are still debated. The problem is not probiotic therapy but rather fermented food products to which small amounts of probiotics are added. After we reviewed the literature, we did not find enough data to safely recommend the use of these products in children receiving chemotherapy (9). Nevertheless, probiotic therapy is recommended for many immunocompromised patients, such as preterm infants and persons with chronic inflammatory bowel disease (10). We believe that this case of B. breve sepsis in an oncology patient underscores the invasive potential of probiotics.

Concordant acute myeloblastic leukemia in monozygotic twins with germline and shared somatic mutations in the gene for CCAAT-enhancer-binding protein α with 13 years difference at onset

Since the first report,[1][1] very few monozygotic pairs of twins with concordant leukemia have been described.[2][2] The interval between the onset of overt disease in each of the twins usually varies from some months to some years.[2][2] Genetic changes in childhood acute leukemia are highly

Rituximab for the treatment of high titre inhibitors in mild haemophilia A.

Dear Sir, The development of allo-antibodies against exogenous “wild-type” factor VIII (FVIII), known as “inhibitors”, is currently the most serious complication in the management of patients with haemophilia A. Inhibitors neutralise FVIII, leading to treatment failure and are, therefore, usually detected when bleeding episodes fail to respond to appropriate FVIII replacement. Although such inhibitors are most frequently seen in patients with severe haemophilia A, the development of antibodies against FVIII in mild haemophilia A can cause considerable clinical problems because the inhibitors, directed against both endogenous and exogenous FVIII, usually change the bleeding phenotype1. We present the case of a 12-year old male with mild haemophilia A (FVIII activity 7%) caused by a mutation in exon 12p. Arg593Cys within the A2 domain in whom successful 4-year long eradication of high-titre inhibitors was achieved by four standard doses of rituximab. The patient urgently needed abdominal surgery because of acute appendicitis with perforation and peritonitis. Before the operation he was treated three times only with DDAVP and antifibrinolytics. The family and personal history was unremarkable except that the patient’s 3-year older brother was diagnosed with diabetes mellitus type 1 at the age of 5 years. Immediately before the surgery the patient was treated with a bolus of recombinant FVIII concentrate (Advate®, Baxter, Lessines, Belgium) and later with a continuous infusion of the same concentrate. On the 6th post-operative day mild intraperitoneal bleeding occurred. On the 8th post-operative day haematochezia appeared, the patient became haemodynamically unstable and computed tomography of the abdomen and pelvis revealed a pelvic fluid collection of 10×10 cm in size. FVIII activity was 38%: a bolus of FVIII concentrate was added and the FVIII activity increased to 87%. Given the severe intrapelvic bleeding, the treatment with FVIII by continuous infusion was continued for another 2 weeks trying to maintain the FVIII activity at 40–50%. In spite of an increase of FVIII consumption FVIII activity decreased from the 23th post-operative day, but inhibitors were still negative (Figure 1). Inhibitors were clearly positive on the 26th post-operative day, when the haematoma had almost cleared and the continuous infusion of FVIII was discontinued. However, four bolus injections of FVIII (36 U/kg/day) were additionally given on post-operative days 27, 29, 32 and 34 with the intention of introducing low-dose immune tolerance induction. The inhibitor titre increased to its highest level, 32 Bethesda units (BU)/mL on the 74th post-operative day (Figure 1). Figure 1 Factor VIII activity in correlation with factor VIII consumption and inhibitor levels. Corticosteroid therapy at a dose of 1 mg/kg, started on day 79 post-operatively, lowered the inhibitor titre in 17 days from 32 to 20 BU/mL. At that time corticosteroid therapy was tapered and inhibitors titre rose again to 30 BU/mL on day 108 post-operatively. On day 112 rituximab was introduced at the standard dose of 375 mg/m2 per week for four weeks. By 4 months after the beginning of the rituximab therapy the titre of inhibitors was steadily decreasing to the level ×0.6 BU/mL and the concentration of FVIII slowly increasing to 4–9%. However, 14 months after the beginning of the rituximab therapy the inhibitor level increased again -but only up to 1.9 BU/mL- and FVIII activity fell to 2%. This situation lasted less than 4 months. Since then inhibitors have not been detectable and FVIII activity has been at the basal level, around 7% (Figure 1). Huge and very painful muscle bleeds occurred with minimal trauma from day 42 until day 151 post-operatively, the period when FVIII activity was below 2%. These bleeds were successfully treated with recombinant human coagulation factor VIIa (NovoSeven®, Novo Nordisk A/S, Copenhagen, Denmark) and tranexamic acid (Cyklokapron®, Meda Manufacturing GmbH, Cologne, Germany). We noted that the patient needed this therapy only as long as he had pain. Later complete resorption of bleeding was achieved, independently of further treatment with NovoSeven. When the inhibitors decreased below 2 BU/mL and FVIII activity rose to at least 2% there were only occasional bleeds which were manageable with subcutaneous or intranasal DDAVP with or without the addition of tranexamic acid. The concentrations of B lymphocytes 2, 7 and 12 months after beginning of the rituximab therapy were decreased (0.0-0.004-0.166×109/L; normal range according to age: 0.2–0.5×109/L). Despite transient B lymphocyte depletion the patient was without infection. At the next B lymphocyte measurement, 44 months after beginning rituximab therapy, their concentration was 0.274×109/L, which is in the normal range for the patient’s age (0.1–0.4×109/L). Despite B-cell recovery an increase in inhibitor concentration was not observed. The management of patients with mild haemophilia A depends on the basal FVIII activity level, the type of bleeding, the type of surgical intervention or invasive procedure and the presence of inhibitors. The development of inhibitors in patients with mild/moderate haemophilia A is a major complication, because the bleeding phenotype of the patient becomes similar to that of a patient with severe or acquired haemophilia A1. Some specific missense mutations, mostly located within the C1 and C2 domains of the light chain and within the A2 domain of the heavy chain of the FVIII molecule, are associated with an unexpectedly high incidence of inhibitors1. Eckhardt et al. collected data from 138 patients with mild/moderate haemophilia A over a 28-year period and identified a 10-fold increased risk of developing inhibitors in patients with the Arg593Cys missense mutation2. In a study by Mauser-Bunschoten et al., age at first exposure, age at peak treatments, number of peak treatments and Arg593Cys mutation were significantly associated with the development of an inhibitor in mild haemophilia A patients, while continuous infusion with FVIII was not3. Our patient had multiple risk factors for developing an inhibitor: type of mutation, major surgery, suggested to facilitate an antibody response against exogenous FVIII, intensive and first exposure to FVIII, and, potentially, continuous infusion of FVIII. Immune tolerance induction (ITI) is the most common approach used to eliminate inhibitors that develop in patients with haemophilia A. Limited data are available on ITI in patients with mild disease and the treatment seems to be less effective than in severe haemophilia A. Dunkley et al. presented preliminary success with the use of rituximab to treat a patient with mild haemophilia A with inhibitors without the use of ITI. The same group of authors proposed a treatment protocol with rituximab for patients with mild haemophilia A with inhibitors4. To our knowledge, according to published data, our patient with mild haemophilia A with inhibitors is the youngest to have been treated with rituximab. Successful, long-term inhibitor eradication was achieved through four standard doses of rituximab. The observation period of 4 years is the longest among published cases5.

Spectrum of F8 gene mutations in haemophilia A patients from Slovenia

1 Liras A, Gaban AS, Rodriguez-Merchan EC. Cartilage restoration in haemophilia: advanced therapies. Haemophilia 2012; 18: 672–9. 2 McAlindon TE, Driban JB, Lo GH. Osteoarthritis year 2011 in review: clinical. Osteoarthritis Cartilage 2012; 20: 197–200. 3 Intema F, Thomas TP, Anderson DD et al. Subchondral bone remodeling is related to clinical improvement after joint distraction in the treatment of ankle osteoarthritis. Osteoarthritis Cartilage 2011; 19: 668–75. 4 Lafeber FP, Intema F, Van Roermund PM, Marijnissen AC. Unloading joints to treat osteoarthritis, including joint distraction. Curr Opin Rheumatol 2006; 18: 519–25. 5 Intema F, Van Roermund PM, Marijnissen AC et al. Tissue structure modification in knee osteoarthritis by use of joint distraction: an open 1-year pilot study. Ann Rheum Dis 2011; 70: 1441–6. 6 Nishino T, Chang F, Ishii T, Yanai T, Mishima H, Ochiai N. Joint distraction and movement for repair of articular cartilage in a rabbit model with subsequent weight-bearing. J Bone Joint Surg Br 2010; 92: 1033– 40. 7 Van Meegeren ME, Van Veghel K, De Kleijn P et al. Joint distraction results in clinical and structural improvement of haemophilic ankle arthropathy: a series of three cases. Haemophilia 2012; 18: 810–7. 8 Lee V, Srivastava A, PalaniKumar C et al. External fixators in haemophilia. Haemophilia 2004; 10: 52–7. 9 van Valburg AA, van Roy HL, Lafeber FP, Bijlsma JW. Beneficial effects of intermittent fluid pressure of low physiological magnitude on cartilage and inflammation in osteoarthritis. An in vitro study. J Rheumatol 1998; 25: 515–20. 10 Roosendaal G, van Rinsum AC, Vianen ME, van den Berg HM, Lafeber FP, Bijlsma JW. Haemophilic arthropathy resembles degenerative rather than inflammatory joint disease. Histopathology 1999; 34: 144–53.

Low-dose continuous infusion of factor VIII in patients with haemophilia A.

BACKGROUND Patients with haemophilia A (HA) or B (HB) can be given prophylactic or on-demand treatment administered by continuous infusion or bolus injections of factor VIII (FVIII) or IX (FIX). In this study we evaluated the efficacy and safety of low-dose continuous infusion of FVIII or FIX. MATERIAL AND METHODS We studied all eligible patients with HA or HB treated with continuous infusion of factor concentrates over an 18-year period in a single Slovenian Haemophilia Comprehensive Care Centre. Treatment started with a bolus injection of FVIII or FIX, followed by continuous infusion at the initial rate of 2 IU/kg/h of FVIII in HA patients and 4.5 IU/kg/h of FIX in HB patients. The infusion rate was subsequently adjusted according to the indication for therapy. RESULTS A total of 66 continuous infusions (40 in major surgery, 10 in minor surgery and 16 with bleeding episode) in 46 HA patients and 16 (15 in severe and 1 in mild HA) in eight HB patients were included in the study. During the first week of treatment, the median continuous infusion rates in HA patients undergoing major surgery, minor surgery and a bleeding event were 2.18 (0.75-3.68), 1.48 (1.0-2.54) and 2.24 (1.33-3.93) IU/kg/h, respectively. The median FVIII activities were 0.69 (0.37-1.19), 0.47 (0.39-0.84) and 0.52 (0.36-1.06) IU/mL. After the first week of treatment, even lower doses of FVIII were needed. Red blood cell transfusions had to be administered to three patients (2 with severe and 1 with moderate HA) during the continuous infusion and inhibitors developed in five patients. In HB patients, the median continuous infusion rate was 1.85 (1.07-2.94) IU/kg/h and the median FIX activity was 0.62 (0.30-1.04) IU/mL. Red blood cell transfusions were not required, and thrombophlebitis and inhibitors did not appear. DISCUSSION Overall, low-dose continuous infusion was shown to be an effective and safe way of treating patients with HA. The protocol used also proved efficient and safe in all HB patients.

PROCALCITONIN AND INTERLEUKIN-6 AS MARKERS OF SEVERE INFECTION IN CHILDREN WITH FEBRILE NEUTROPENIA

Background. The results of the study conducted to determine whether procalcitonin (PCT) and interleukin-6 (IL-6) are more sensitive and specific markers of severe infection in children with febrile neutropenia (FN) than routinelly used C-reactive protein (CRP) are presented in the article. 68 episodes of FN experienced by 32 patients were divided into three groups according to the site of infection. Group 1: episodes of bacteraemia and/or clinical sepsis (n = 16), group 2: episodes of focal infection (n = 16) and group 3: episodes of fever of unknown origin (FUO) (n = 36). Blood samples for further PCT and IL-6 determination were collected on three consecutive days. CRP concentrations were measured daily in each patient until the resolution of fever. PCT, IL-6 and CRP concentrations were measured on one occassion in each of the 18 afebrile patients with malignant disase forming the reference group. Serum PCT and IL-6 concentrations were measured by immunochemiluminometric and immunoenzymatic assay. Receiver Operating Characteristic (ROC) curves were used to determine optimum sensitivity, specificity, positive and negative predictive values and diagnostic accuracy of the studied parameters. Conclusions. PCT and IL-6 were found to be earlier and more sensitive markers of severe infection in neutropenic patients than CRP. The erliest one was IL-6, followed by PCT and CRP. Sequential determination of PCT up to 72 hours improved its diagnostic value, which was not the case for IL-6.In patients with gramnegative bacteraemias PCT concentracions were 3–5 times higher comparing to grampositive, whereas IL-6 concentrations were comparable in both groups.