Gábor Kovács

ABCC1 polymorphisms in anthracycline-induced cardiotoxicity in childhood acute lymphoblastic leukaemia

Anthracyclines are potent cytostatic drugs, the correct dosage being critical to avoid possible cardiac side effects. ABCC1 [ATP‐binding cassette, sub‐family C, member 1; also denoted as MRP1 (multidrug resistance‐associated protein 1)] is expressed in the heart and takes part in the detoxification and protection of cells from the toxic effects of xenobiotics, including anthracyclines. Our objective was to search for associations between LV (left ventricular) function and single‐nucleotide polymorphisms of the ABCC1 gene in children receiving anthracycline chemotherapy. Data of 235 paediatric patients with acute lymphoblastic leukaemia was analysed. Patients were followed‐up by echocardiography (median follow‐up 6.3 years). Nine polymorphisms in the ABCC1 gene were genotyped. The ABCC1 rs3743527TT genotype and rs3743527TT—rs246221TC/TT genotype combination were associated with lower LVFS (left ventricular fractional shortening) after chemotherapy. The results suggest that genetic variants in the ABCC1 gene influence anthracycline‐induced LV dysfunction.

Association of some rare haplotypes and genotype combinations in the MDR1 gene with childhood acute lymphoblastic leukaemia

To investigate their possible roles in disease susceptibility and some disease characteristics we genotyped C3435T and G2677T/A polymorphisms in multidrug resistance-1 (MDR1) gene with a single base extension method and the G34A and C421A polymorphisms of the breast cancer resistance protein gene with an allelic discrimination system in 396 children with acute lymphoblastic leukaemia (ALL) and 192 control patients. While the distribution of individual alleles and genotypes did not differ between patients and controls, there were significant differences in the frequencies of some rare haplotypes and genotype combinations in the MDR1 gene between the two groups.

Synergistic interaction of ABCB1 and ABCG2 polymorphisms predicts the prevalence of toxic encephalopathy during anticancer chemotherapy.

Polymorphisms of the ABCB1 (MDR1) and ABCG2 (BCRP) genes were reported to alter the expression and function of these drug transporters. Both proteins are present at the main pharmacokinetic barriers including the blood–brain barrier. Data from 291 children with acute lymphoblastic leukaemia were analysed in this retrospective study. ABCB1 3435T>C, 2677G>T/A, 1236C>T and ABCG2 421C>A, 34G>A genotypes were determined. Encephalopathy episodes were more frequent among those with ABCB1 3435TT genotype than in the 3435CC/CT group (odds ratio (OR) 3.5; P=0.03). Patients with the ABCG2 421A allele tended to have more complications than wild type homozygotes (OR=2.0; P=0.25). The rate of the adverse effect was similar in those harbouring no or only one of the predisposing genotypes, that is, either ABCB1 3435TT or ABCG2 421AA/AC. However, significantly more children suffered encephalopathy in the group with both predisposing genotypes (OR=12.3; P=0.005). In conclusion, these variations exert synergistic effect in predisposing patients to toxic neurological complications of chemotherapy.

Comparison of pharmacokinetics and toxicity after high-dose methotrexate treatments in children with acute lymphoblastic leukemia

We carried out a detailed comparative study of the pharmacokinetics and toxicity of methotrexate (MTX) and 7-hydroxy-methotrexate (7-OH-MTX) after high-dose intravenous methotrexate (HD-MTX) in children with acute lymphoblastic leukemia (ALL). Overall, 65 children were treated with 5 g/m2/24 h MTX and 88 children were treated with 2 g/m2/24 h MTX according to ALL-BFM 95 and ALL IC-BFM 2002 protocols (mean age: 6.4 years, range 1.0–17.9 years). A total of 583 HD-MTX courses were analyzed. Serum MTX and 7-OH-MTX levels were measured at 24, 36, and 48 h, and cerebrospinal fluid (CSF) MTX levels were determined 24 h after the initiation of the infusion. The area under the concentration–time curve was calculated. Hepatotoxicity, nephrotoxicity, and bone marrow toxicity were estimated by routine laboratory tests. We investigated pharmacokinetics and toxicity in distinct age groups (<6 and >14 years). 5 g/m2/24 h treatments resulted in higher serum and CSF MTX and 7-OH-MTX levels (P<0.05). The CSF penetration rate of MTX was independent of the MTX dose [2.3% (95% confidence interval: 1.7–2.5%) vs. 2.8% (95% confidence interval: 2.4–3%)]. The CSF MTX concentration was correlated with the 24 h MTX serum level (r=0.38, P<0.0001). Repeated treatments did not alter MTX or 7-OH-MTX levels. 7-OH-MTX levels were correlated with nephrotoxicity (r=0.36, P<0.0001). Higher MTX levels and toxicity occurred more frequently in children aged older than 14 years (P<0.05). Therapeutic serum and CSF MTX concentrations can be achieved more reliably with 5 g/m2/24 h treatments. To predict the development of toxicity, monitoring of the level of the 7-OH-MTX is useful. Monitoring of pharmacokinetics is essential to prevent the development of severe adverse events in adolescents.

Rheumatic symptoms in childhood leukaemia and lymphoma-a ten-year retrospective study

BackgroundThe initial symptoms of childhood leukaemia and lymphoma are often similar to those of juvenile idiopathic arthritis (JIA). In our study, we analyzed the frequency and characteristics of musculoskeletal complaints as the initial presenting symptoms of newly diagnosed leukaemia and lymphoma patients in the past 10xa0years in our clinic.MethodsUsing the Hungarian Tumour Register, we performed a retrospective analysis of the medical records of 166 new leukaemia and 95 new lymphoma pediatric patients treated from 1999 to 2009 at the 2nd. Dept. of Paediatrics of the Semmelweis University in Budapest.ResultsTwenty percent of the leukaemic (33 children) and 2% of the lymphoma patients (2 children) had musculoskeletal symptoms at first presentation. Two-thirds of both groups of patients had other general symptoms like fever and/or fatigue. The hip was the most frequently affected joint (7/33) in the leukaemic patients. Twenty-four percent of all the children had been previously evaluated by an orthopaedist; 12% had visited another rheumatologist prior to diagnosis. Imaging had been done in an unexpectedly low number of patients prior to referral to our unit (radiographs: 16 or 48%, ultrasound: 5 patients or 15%). Radiographs of the affected joints were abnormal in only one case (1/16, 6%). The joint ultrasound was abnormal in only three children of 5 studied (3/5, 60%). Anaemia (26/32, 6%), thrombocytopenia (78%) and LDH elevation (3–4 times the normal count) were frequent in the leukaemic patients. Half of the cases had a normal leukocyte count. The lymphoma group had similar results. Two patients of the leukaemia group received steroid treatment before the final diagnosis. Severe pain out of proportion to physical findings is another clue.ConclusionsHaematologic malignancies must be excluded before initiation of therapy for childhood arthritis among children presenting with musculoskeletal signs and symptoms, particularly in atypical cases. Malignancies are to be suspected when pain is disproportionately severe compared to the physical examination findings, and when anaemia, thrombocytopenia, and an elevated LDH level are present. Diagnosing leukaemia early is important because the use of steroids and immunosuppressive medications may mask and delay its diagnosis. Additionally, pre-treatment of presumed JIA patients with these drugs who eventually are diagnosed to have a malignancy may lead to the malignancy being steroid-resistant and more difficult to treat.

The glucocorticoid receptor gene polymorphism N363S predisposes to more severe toxic side effects during pediatric acute lymphoblastic leukemia (ALL) therapy

The survival rates in childhood acute lymphoid leukemia (ALL) have improved dramatically; however, patients still suffer from a variety of drug-related toxicities. Individualized therapy regimens promise the least toxic therapy regimen with the best hematologic outcome. Our aim was to investigate whether increased individual glucocorticoid sensitivity due to the N363S polymorphism of the glucocorticoid receptor increased susceptibility to steroid-related toxicities during ALL therapy. A total of 346 pediatric ALL patients were involved in the present study. N363S carrier status was investigated by allele-specific PCR. Clinical and laboratory signs of glucocorticoid-related toxicities, Day 8 prednisone response, and 5-year event-free survival were analyzed and compared retrospectively. Thirty-two of the 346 patients were heterozygous carriers (9.2xa0%). Hepatotoxicity (31.3 vs. 11.2xa0%, pxa0=xa00.004, carriers and non-carriers, respectively) and glucose metabolism abnormalities (18.8 vs. 3.8xa0%, pxa0=xa00.001, carriers and non-carriers, respectively) were significantly more frequent among carriers. There was no difference in the incidence of hypertension and encephalopathy/psychosis among carriers and non-carriers. Carriers were also more prone to have a combination of toxicities. All 363S carriers were good prednisone responders (100xa0%) and had significantly better 5-year event-free survival rates (93.1 vs. 71.86xa0%, pxa0=xa00.012), whereas among non-carriers there were more poor prednisone responders (8.28xa0%) and worse 5-year event-free survival rates. Patients with the N363S polymorphism in the glucocorticoid receptor are more prone to steroid-related toxicity during ALL therapy and should be monitored more closely. Patients with N363S polymorphism of the glucocorticoid receptor may be appropriate candidates for inclusion in the design of individualized therapies.

Late Immune Recovery in Children Treated for Malignant Diseases

In this study we analyzed the recovery of the immune system in children after completion of the therapy. We analysed 88 children (51 boys, 37 girls, mean age at diagnosis: 7.8xa0years) receiving chemotherapy for malignant diseases (43 acute lymphoblastic leukemia, 15 lymphoma, 20 bone tumor, ten other solid tumors). Serum immunoglobulin levels (Ig), natural killer activity (NK), antibody-dependent cellular cytotoxicity (ADCC) and T and B cell proliferation were determined 1xa0year after cessation of therapy. The mean levels of Ig were in the normal range at a mean of 13xa0months after chemotherapy (IgG: 11.2u2009±u20093.3, IgA: 1.6u2009±u20090.9, IgM: 1.0u2009±u20090.5xa0g/l), however in the leukemic patients serum IgG was below the lower limit of the normal range in 3/43 (7.0%) cases, serum IgA was low in 5/43 (11.6%) and serum IgM was decreased in 4/43 (9.3%) cases. In the solid tumor patients IgG values were within the normal range and only 2–2/45 children had lower values for IgA and IgM (4.4%). NK activity decreased in 7/43 (16.3%) leukemic patients, and in 3/45 (6.7%) solid tumor patients, ADCC decreased in 8/43 (18.6%) and 3/45 (6.7%), respectively (pu2009<u20090.001). B-cell blastic transformation was decreased in 3/43 (7%) leukemic patients and in 4/45 (8.9%) solid tumor patients. At the same time T-cell blastic transformation was altered in 5/43 (11.6%) and in 4/45 (8.9%) cases, respectively. Leukemic patients had significantly more infections during the first year after chemotherapy than solid tumor patients (1.60u2009±u20091.18 vs 0.96u2009±u20091.14; pu2009=u20090.011). No significant correlations could be found between the investigated immune parameters and the number and severity of infections. It is concluded, that cytotoxic therapy can lead to long-term depression of the immune system, first of all in leukemic patients.

Associations of novel genetic variations in the folate-related and ARID5B genes with the pharmacokinetics and toxicity of high-dose methotrexate in paediatric acute lymphoblastic leukaemia.

High‐dose methotrexate (HD‐MTX) plays an important role in the consolidation therapy of acute lymphoblastic leukaemia (ALL) in many treatment regimens worldwide. However, there is a large interpatient variability in the pharmacokinetics and toxicity of the drug. We investigated the influence of single nucleotide polymorphisms (SNPs) in genes of the folate metabolic pathway, transporter molecules and transcription proteins on the pharmacokinetics and toxicity of MTX and 7‐hydroxy‐methotrexate (7‐OH‐MTX). 63 SNPs of 14 genes were genotyped and a total of 463 HD‐MTX courses (administered according to the ALL‐BFM 95 and ALL IC‐BFM 2002 protocols) were analysed. Haematological, hepatic and renal toxicities, estimated by routine laboratory parameters were evaluated. Random forest and regression trees were used for variable selection and model building. Linear mixed models were established to prove the significance of the selected variables. SNPs (rs4948502, rs4948496, rs4948487) of the ARID5B gene were associated with the serum levels of MTX (P < 0·02), serum levels and area under the curve of 7‐OH‐MTX (P < 0·02) and with hypoproteinaemia (P = 0·004). SLCO1B1 rs4149056 also showed a significant association with serum MTX levels (P < 0·001). Our findings confirm the association of novel genetic variations in folate‐related and ARID5B genes with the serum MTX levels and acute toxicity.

Subacute cardiotoxicity caused by anthracycline therapy in children: can dexrazoxane prevent this effect?

Anthracyclines are potent cytostatic drugs, but their use may be limited by cardiac toxicity. In the study reported here, we investigated the incidence and outcome of anthracycline-induced cardiotoxicity and the possible preventive role of dexrazoxane. A total of 108 patients with the diagnosis of osteosarcoma, Ewing-sarcoma, soft tissue sarcoma and leukemia were studied between 1991 and 2003. Of these, 41 children (treated from 1996) received dexrazoxane (Group D) as chemotherapy from the beginning of their treatment (mean follow-up: 8.2±3.9 years). The dose of dexrazoxane was 20-fold higher than the anthracycline dose and was administered as a 20-min sodium-lactate infusion. The control (Group C) cohort comprised 67 children (all treated before 1996) who did not receive any cardioprotection (mean follow-up: 12.3±3.2 years). All patients in the C and D groups received anthracyclines in the form of short infusions (1–3 h). The cumulative dose of anthracycline did not differ between the two groups (Table 1). Supportive treatment did not change during the investigated period. Cardiac ultrasound examinations were performed regularly, and fractional shortening (FS) for detecting left ventricular function was measured. The incidence of acute cardiotoxicity was 13.4% in Group C patients and 7.3% in Group D patients, a nonsignificant difference. Cardiac function, expressed as FS, was abnormally low (<30%) 2 years after therapy in 13.7% of the Group C patients and in 0% of the Group D patients (p=0.034) (Fig. 1a) and was still abnormally low 3 years after therapy in 19.3% of the Group C patients and 0% of the Group D patients (p=0.008) (Fig. 1b). At the 5year follow-up, FS was under 30% in 14.9% of the Group C patients and 2.4% of the Group D patients (Fig. 1c). Seven patients in Group C needed heart-protective drugs (digitalis, furosemide, angiotensine converting enzyme inhibitors) for congestive heart failure for at least 6 months. Only one patient in Group D received cardiac drugs. Dilatative cardiomyopathy is a severe complication of anthracycline therapy, and even in children, lower cumulative doses (<300 mg/m 2 ) may cause irreversible congestive heart failure [3]. The incidence of slight cardiotoxicity has been reported to vary between 12 and 32% and mortality or severe dysfunction to vary between 2 and 7%, depending

Clinical relations of methotrexate pharmacokinetics in the treatment for pediatric osteosarcoma.

PurposeHigh-dose methotrexate (HD-MTX) with leucovorin rescue is widely used to treat osteosarcoma. Our objectives were to assess correlations between pharmacokinetic parameters and the outcome of osteosarcoma and to analyze the relation between HD-MTX exposure and toxicity.MethodsPharmacokinetic data of 105 patients with osteosarcoma treated with 989 HD-MTX courses were evaluated. Pharmacokinetic parameters (clearance, half-life and AUC) were calculated based on methotrexate (MTX) serum levels measured at 6, 24, 36, 48xa0h after the initiation of the infusion. Clinical data were collected by retrospective chart review. Hepato-, nephro- and bone marrow toxicity parameters were categorized according to Common Toxicity Criteria v.3.0, and MTX dose intensity was calculated. Event-free survival (EFS) and overall survival (OS) were estimated according to the Kaplan–Meier method.ResultsPatients with serious hepatotoxicity had higher mean peak MTX concentrations (pxa0<xa00.0001), 24-h (pxa0=xa00.001) and 48-h MTX serum levels (pxa0=xa00.008) and AUC0–48 (pxa0<xa00.0001), and lower MTX clearance (pxa0=xa00.0002). No significant association was found between toxicity and age, gender, presence of metastases or histological tumor response. Patients with higher 48-h MTX serum levels had significantly better OS and EFS. Higher dose intensity was associated with better EFS (pxa0=xa00.0504). There was no association between presence of toxicity and survival.ConclusionThere was correlation between MTX exposure and the incidence of toxicity. Higher serum concentrations at 48xa0h were associated with a better 5-year OS and EFS. These results suggest that higher MTX exposure may lead to serious side effects, but it also improves treatment outcome.