Digital gene expression profiling of primary acute lymphoblastic leukemia cells.

We determined the genome-wide digital gene expression (DGE) profiles of primary acute lymphoblastic leukemia (ALL) cells from 21 patients taking advantage of 'second-generation' sequencing technology. Patients included in this study represent four cytogenetically distinct subtypes of B-cell precursor (BCP) ALL and T-cell lineage ALL (T-ALL). The robustness of DGE combined with supervised classification by nearest shrunken centroids (NSC) was validated experimentally and by comparison with published expression data for large sets of ALL samples. Genes that were differentially expressed between BCP ALL subtypes were enriched to distinct signaling pathways with dic(9;20) enriched to TP53 signaling, t(9;22) to interferon signaling, as well as high hyperdiploidy and t(12;21) to apoptosis signaling. We also observed antisense tags expressed from the non-coding strand of ~50% of annotated genes, many of which were expressed in a subtype-specific pattern. Antisense tags from 17 gene regions unambiguously discriminated between the BCP ALL and T-ALL subtypes, and antisense tags from 76 gene regions discriminated between the 4 BCP subtypes. We observed a significant overlap of gene regions with alternative polyadenylation and antisense transcription (P<1 × 10(-15)). Our study using DGE profiling provided new insights into the RNA expression patterns in ALL cells.

The frequency and prognostic impact of dic(9;20)(p13.2;q11.2) in childhood B-cell precursor acute lymphoblastic leukemia: results from the NOPHO ALL-2000 trial.

The dic(9;20)(p13.2;q11.2) is reported to be present in ∼2% of childhood B-cell precursor acute lymphoblastic leukemia (BCP ALL). However, it easily escapes detection by G-banding analysis and its true prevalence is hence unknown. We performed interphase fluorescence in situ hybridization analyses-in a three-step manner-using probes for: (i) CDKN2A at 9p21, (ii) 20p and 20q subtelomeres and (iii) cen9 and cen20. Out of 1033 BCP ALLs diagnosed from 2001 to 2006, 533 were analyzed; 16% (84/533) displayed 9p21 deletions, of which 30% (25/84) had dic(9;20). Thus, dic(9;20)-positivity was found in 4.7% (25/533), making it the third most common genetic subgroup after high hyperdiploidy and t(12;21)(p13;q22). The dic(9;20) was associated with a female predominance and an age peak at 3 years; 18/25 (72%) were allocated to non-standard risk treatment at diagnosis. Including cases detected by G-banding alone, 29 dic(9;20)-positive cases were treated according to the NOPHO ALL 2000 protocol. Relapses occurred in 24% (7/29) resulting in a 5-year event-free survival of 0.69, which was significantly worse than for t(12;21) (0.87; P=0.002) and high hyperdiploidy (0.82; P=0.04). We conclude that dic(9;20) is twice as common as previously surmised, with many cases going undetected by G-banding analysis, and that dic(9;20) should be considered a non-standard risk abnormality.

Bone marrow fibrosis in childhood acute lymphoblastic leukemia correlates to biological factors, treatment response and outcome.

We retrospectively evaluated reticulin fiber density (RFD) in 166 diagnostic bone marrow (BM) biopsies and 62 biopsies obtained at treatment day 29 from children with acute lymphoblastic leukemia (ALL). Patients with B-cell precursor (BCP)-ALL showed higher RFD as compared to patients with T-cell ALL (P<0.001). RFD correlated negatively with white blood cell count (P=0.008) in BCP-ALL patients. Patients with high-hyperdiploid ALL (51-61 chromosomes), no high-risk criteria and low RFD showed a favorable outcome when compared to similar patients with high RFD (P=0.002). In BCP-ALL patients, RFD at diagnosis correlated to the levels of minimal residual disease (MRD) analyzed by flow cytometry on treatment day 29 (P=0.001). Accordingly, patients with MRD > or = 10(-4) presented higher RFD at diagnosis compared to patients with MRD < 10(-4) (P=0.003). BCP-ALL patients with low RFD at diagnosis and a rapid reduction of RFD on day 29 had a favorable outcome compared to patients with the same baseline RFD level at diagnosis but a slow RFD reduction (P=0.041). To our knowledge, these findings are novel and may indicate BM fibrosis as a new valuable prognostic marker in childhood ALL. Expanded use of BM biopsy both at diagnosis and during follow-up is suggested.

Dendritic cell regeneration in the bone marrow of children treated for acute lymphoblastic leukaemia.

Dendritic cells (DC) play a pivotal role in coordinating functions of the immune system. Little is known about DC levels in the bone marrow (BM) of patients receiving cytostatic treatment. We investigated DC levels by flow cytometry in BM at diagnosis, during and post-treatment in 76 children with acute lymphoblastic leukaemia (ALL). The levels of both plasmacytoid DC (pDC) and myeloid DC (mDC) were profoundly reduced at diagnosis. However, the levels of pDC and mDC were significantly higher in T-precursor ALL patients when compared with B-precursor ALL patient group (P = 0.044 and 0.041 respectively). Both subsets normalized in both standard-risk (SR) and high-risk patients after the end of induction at day 50. Patients with minimal residual disease (MRD) at day 50 had significantly higher pDC levels than MRD-negative patients (P = 0.021). In B-precursor SR ALL patients, mDC levels but not pDC levels decreased during prolonged maintenance treatment, remaining reduced at the end of treatment (P = 0.032) and at 6 months post-treatment (P = 0.028). In conclusion, levels of DC in BM normalize quickly in children treated for ALL. Long-term treatment may more profoundly affect mDC subset, which shows reduced levels several months after treatment.

Cellular drug sensitivity in MLL-rearranged childhood acute leukaemia is correlated to partner genes and cell lineage.

Rearrangements in the 11q23 region, the site of the mixed lineage leukaemia (MLL) gene, are found in both childhood acute myeloid (AML) and lymphoblastic (ALL) leukaemia. We studied the in vitro drug resistance by the fluorometric microculture cytotoxicity assay (FMCA) in 132 children with AML and 178 children with ALL (aged 0-17 years). In AML, children with t(9;11) (n = 10) were significantly more sensitive to cytarabine (P < 0.001) and doxorubicin (P = 0.005) than non-11q23 rearranged patients (n = 108). Children with other 11q23 rearrangements (n = 14) differed less from non-rearranged children. The 'AML-profile' common to all three groups included relative resistance to glucocorticoids and vincristine. In ALL, children with 11q23 rearrangement (n = 22) were significantly more sensitive to cytarabine (P = 0.026) than children without 11q23 rearrangement (n = 156), also after stratification for white blood cell count. In conclusion, the findings indicate that the cellular drug resistance is correlated to both the cell lineage and the type of 11q23 rearrangement. High cellular sensitivity to cytarabine and doxorubicin might explain the excellent treatment results in children with AML and t(9;11). The present study supports the strategy of contemporary protocols to include high-dose cytarabine in the treatment of 11q23-positive patients both in AML and ALL.

Pulmonary function after autologous bone marrow transplantation in children: a long-term prospective study.

We performed serial pulmonary function tests (PFTs) consisting of spirometry and diffusing capacity in 26 children after BMT. The median follow-up was 10 years. The influence of total body irradiation (TBI) on long-term pulmonary function was of particular interest. In the 20 children who had received TBI, after an initial decrease the PFTs showed recovery, but the mean lung volumes were still significantly decreased 5 years after BMT at 10% below baseline. The proportions of children with restrictive impairment 5 and 10 years after BMT were 20 and 21%, respectively. Only one child was diagnosed with obstructive impairment. The proportions of children with isolated diffusing impairment at 5 and 10 years were 7/20 (35%) and 7/13 (54%), respectively. Six children had received chemotherapy only and showed isolated diffusing impairment as the only long-term sequela in 4/5 and 1/3 at 5 and 10 years. Our main finding was that there was little change in PFTs 1-10 years after BMT. TBI was associated with persistently decreased lung volumes in a proportion of patients, whereas chemotherapy also might have been of importance for the development of impaired gas exchange.

Pubertal development and final height after autologous bone marrow transplantation for acute lymphoblastic leukemia.

We describe pubertal development and growth in 17 children who underwent bone marrow transplantation (BMT), including total body irradiation (TBI) for ALL. Seven children also received cranial irradiation (CI) and five boys testicular irradiation. All underwent transplantation before (n=15) or at the beginning of (n=2) puberty and reached a final height (FH). Puberty started spontaneously in all boys not given testicular irradiation. All boys who received testicular irradiation developed hypergonadotrophic hypogonadism. Puberty started spontaneously in two girls and was induced with increasing doses of ethinylestradiol in two girls. In two girls, a low dose of ethinylestradiol was given until menarche. In one girl with early onset of puberty and short stature, puberty was blocked with a GnRH analogue. The standard deviation score for height decreased significantly from BMT to FH, both in the children who received TBI only (-1.1, P=0.005) as well as in those given additional CI (-1.7, P=0.027). Most of the loss occurred during puberty. In all, 10 children received growth hormone (GH) treatment. CI, young age at BMT, and short duration of GH treatment were predictors of height loss after BMT. Although limited by the small and heterogeneous sample, our study supports the use of early GH treatment in children with decelerating growth rate and low GH levels.

Vincristine in childhood leukaemia: no pharmacokinetic rationale for dose reduction in adolescents.

AIM: To investigate whether there is any pharmacokinetic rationale for the common practice of administering vincristine to adolescents at relatively lower doses than those to younger children. METHODS: A total of 98 children, aged 1.3-17.3 y, with acute lymphoblastic leukaemia (ALL) were studied on day 1 of induction therapy. Plasma samples were drawn before and 10, 30, 360 and 1380 min after injection of vincristine 2.0 mg/m2 (maximum dose 2.0 mg) and analysed by high-performance liquid chromatography. RESULTS: The median value (and range) for distribution half-life was 6.4 min (0.8-11.8), elimination half-life 1014 min (258-2570), volume of distribution 445 L/m2 (137-1241) and total body clearance 362 ml/min/m2 (134-2553). No correlation was found between age and any of these pharmacokinetic parameters. The area under the concentration time curve (AUC) was significantly correlated to age (p = 0.002; p - 0.31), as expected from the dosage of vincristine. The lower AUC in children with a body surface area > 1 m2, which is reached at 8-9 y of age, indicates that they received a less intense treatment because of the capping of the vincristine dose at 2.0 mg. CONCLUSIONS: Vincristine pharmacokinetics were not age dependent in this paediatric population. Thus, we found no pharmacokinetic rationale for dose reduction in adolescents. The common practice of limiting the vincristine dose to 2.0 mg should be carefully reconsidered.

High-dose methotrexate: on the relationship of methotrexate elimination time vs renal function and serum methotrexate levels in 1164 courses in 264 Swedish children with acute lymphoblastic leukaemia (ALL).

PURPOSE: The objectives of the present study were to determine the relationship between methotrexate (MTX) elimination time and various aspects of renal function and to evaluate the prognostic value of elevated serum MTX and creatinine for delayed MTX elimination. PATIENTS AND METHODS: The majority of the 264 children were being treated for ALL. According to the NOPHO-92 protocol, 5 or 8 g MTX/m(2) was administered over 24 h. Serum creatinine was assessed daily. In 11 patients from one centre, renal function was studied in more detail using serum cystatin C, iohexol clearance, and urinary albumin, IgG and protein HC. RESULTS: Increased serum creatinine correlated significantly with the elimination time of MTX, whereas no indications were found of tubular or barrier function damage. Of the 1164 courses, 44 had delayed elimination of MTX (>/=120 h). Serum MTX >150 microM at the end of infusion had a sensitivity of 0.27 and a specificity of 0.94 to predict delayed MTX elimination, and >/=50% increase in serum creatinine during the first treatment day (creatinine ratio) had a sensitivity of 0.32 and a specificity of 0.99. The corresponding risk ratios were 5 and 19 for MTX >150 micro M and creatinine ratio, respectively. In courses with a normal elimination time (<72 h), 99% of the courses had a rise in serum creatinine of less than 50%. CONCLUSIONS: Elevation of serum creatinine by more than 50% is a better predictor of delayed elimination than the level of serum MTX at the end of MTX infusion, especially if information on previous creatinine measurements is used to reduce the impact of an occasionally low serum creatinine value before the start of the MTX infusion.

Skin infection caused by Mycobacterium szulgai after allogeneic bone marrow transplantation.

Infections are responsible for a large part of the morbidity and mortality after BMT because of the sustained impairment of host defenses. We report a case of cutaneous infection caused by Mycobacterium szulgai in a boy who underwent BMT with marrow from a matched unrelated donor.

In vitro activity of the novel cytotoxic agent CHS 828 in childhood acute leukemia.

CHS 828, a pyridyl cyanoguanidine, is a new drug candidate now in phase I/II trials, that has shown promising anticancer activity in experimental tumor models and primary cultures of cancer cells from patients. In this study the fluorometric microculture cytotoxicity assay was used for evaluation of CHS 828 in primary cell cultures from children with acute leukemia. The activity of and interaction with the standard drugs, doxorubicin, melphalan, etoposide and cytosine arabinoside (Ara-C), were also assessed. Samples from 65 patients, 42 with acute lymphocytic leukemia (ALL) and 23 with acute myelocytic leukemia (AML) were tested with 72-h continuous drug exposure. There was 50% cell kill at very low CHS 828 concentrations; median IC50 was 0.01 microM in ALL and 0.03 in AML samples (NS) with large interindividual variability in both groups. ALL samples were significantly more sensitive than AML samples to melphalan, doxorubicin and etoposide, but not to Ara-C. In AML samples, combinations between CHS 828 and each of the four standard drugs resulted in significantly lower cell survival than either drug alone. This was also observed in ALL samples, except for Ara-C. Using the additive interaction model, CHS 828 showed a synergistic effect with melphalan in 67%, doxorubicin in 47%, etoposide in 38% and Ara-C in 14% of AML samples. In most ALL samples subadditive effects were found. Further exploration of CHS 828 in childhood leukemia is warranted, especially in AML.

Pharmacokinetics of doxorubicin in children with acute lymphoblastic leukemia: multi-institutional collaborative study.

BACKGROUND: In adults, it has been shown that the pharmacokinetics of doxorubicin are highly variable, despite standardization of the dose based on body surface area (BSA). The purpose of this study was to determine the plasma concentrations of doxorubicin and its active metabolite doxorubicinol in children treated for acute lymphoblastic leukemia (ALL). PROCEDURE: Children, 107 in number, aged 1.3-17.3 years, were studied at Day 1 of induction therapy according to the current Nordic protocol. Five infants, 3-9 months old, were also included. Plasma samples were drawn 23 hr after the start of a 24-hr infusion of doxorubicin 40 mg/m(2), and analyzed by reversed-phase liquid chromatography. RESULTS: There was a more than 10-fold difference between patients in dose normalized plasma concentration of doxorubicin, median 62.8 ng/ml, range 22.6-334 ng/ml. The doxorubicin concentrations differed significantly between age groups (P = 0.003). Children aged 4-6 years had the highest doxorubicin concentrations, median 77.9 ng/ml, followed by 2-4-year-old children, median 64.3 ng/ml. Both younger and older children had median values of about 50 ng/ml. Patients with white blood cell (WBC) count > 50 x 10(9)/L at diagnosis had significantly lower doxorubicin concentrations, median 55.3 ng/ml, than those with WBC count < 10 x 10(9)/L, median 64.4 ng/ml (P = 0.015). There was no difference in doxorubicin concentration between boys and girls. No correlation was found between doxorubicin levels and serum aminotransferases or serum creatinine. The concentration of doxorubicinol was 13% (median value) of that of doxorubicin. Four infants, 7-9 months old, had plasma clearance between 350-431 ml/min/m(2), which is in the same range as in older children. A 3-month-old infant had a clearance of 181 ml/min/m(2). CONCLUSIONS: The age groups who had the highest doxorubicin concentrations, (2-) 4-6-year-old children, are known to make up a large proportion of standard risk ALL cases with good prognosis. The correlation between doxorubicin plasma levels and clinical effect needs further study. The influence of age, body composition, and tumor burden on the pharmacokinetics of antineoplastic drugs should also be further explored, aiming at improvements in the current dosing regimen based on BSA.

Renal function after autologous bone marrow transplantation in children: a long-term prospective study.

We measured glomerular filtration rate (GFR), effective renal plasma flow (ERPF) and the concentrating capacity of the kidneys in children after autologous BMT. Twenty-six patients had received TBI in their conditioning regimen and 14 patients had received chemotherapy only. Median follow-up was 10 years. Mean GFR before BMT was close to normal in both groups. Mean GFR decreased from 124 [CI 114,134] ml/min/1.73 m(2) before BMT to 99 [CI 82,115] ml/min/1.73 m(2) 6 months after BMT in the + TBI group (P < 0.001). There was no significant change in the -TBI group. Mean ERPF before BMT was high: 1110 [95% CI 830,1390] ml/min/1.73 m(2) in the + TBI group and 910 [CI 570,1250] ml/min/1.73 m(2) in the - TBI group. Six months after BMT, there was a tendency to a decrease in ERPF in the +TBI group, to 760 [CI 580,940] ml/min/1.73 m(2) (P = 0.064). After this initial decrease, GFR and ERPF remained essentially unchanged in both groups. The mean concentrating capacity of the kidneys was normal before and after BMT. In seven patients chronic renal impairment developed after BMT (GFR <70 ml/min/1.73 m(2)). All had received TBI. They had also received more nephrotoxic antibiotics than the other patients. We conclude that TBI was the principal cause of deterioration of renal function after BMT, possibly by limiting compensatory hyperperfusion and resulting in a fall in GFR. Antibiotic treatment may have contributed.

No disadvantage in outcome of using matched unrelated donors as compared with matched sibling donors for bone marrow transplantation in children with acute lymphoblastic leukemia in second remission.

PURPOSE: We evaluated the outcome of children with acute lymphoblastic leukemia (ALL) in second remission (2CR), comparing bone marrow transplantation (BMT) using either matched sibling donors or unrelated donors (URDs). PATIENTS AND METHODS: A total of 65 patients, aged 2 months to 20 years at BMT, with ALL in 2CR underwent allogeneic BMT at seven Nordic centers during 1990 to 1997. Of the first relapses, 85% were in bone marrow; 46% occurred on therapy, and 54%, off therapy. The preparative regimens were cyclophosphamide plus total-body irradiation +/- antithymocyte/antilymphocyte globulin, busulfan plus cyclophosphamide +/- antithymocyte/antilymphocyte globulin, or cytarabine plus total-body irradiation. Of the allografts, 37 were from HLA-matched siblings and 28 were from URDs. RESULTS: In the sibling versus URD graft recipient groups, the posttransplantation 5-year event-free survival was 39% versus 54% (P =.4), the estimated posttransplantation relapse rate was 76% versus 40% (P = not significant [NS]), and the toxic death rate was 19% versus 11% (P = NS). The incidence of significant (grade 2 to 4) acute graft-versus-host disease (GVHD) was 38% versus 64% (P <.05) and was 14% versus 32% (P <.10) for severe (grade 3 to 4) acute GVHD; the incidence of chronic GVHD was 26% versus 57% (P <.05) and was 13% versus 22% (P = NS) for extensive chronic GVHD in the sibling and URD groups. CONCLUSION: BMT with matched URD allografts offers at least equal survival for children with ALL in 2CR, as compared with allografts from matched sibling donors. URD allografts were not associated with a higher toxic mortality rate, although both acute and chronic GVHD were more frequent with URD. Indications for using matched URD allografts in ALL 2CR can be considered the same as for using matched sibling donors.

Androgen-linked control of carbonic anhydrase III expression occurs in rat perivenous hepatocytes; an immunocytochemical study.

Carbonic anhydrase (CA) isozymes CAII and CAIII were assayed by a radioimmunosorbent technique in liver cytosolic fractions and in isolated hepatocytes of adult male and female rats. Male livers contained 0.16 mg of CAII and 57 mg of CAIII per g cytosolic protein. Corresponding values for female livers were 0.34 mg CAII and 4 mg CAIII. Similar values and differences between CAII and III were found in isolated hepatocytes. Neonatal and adult castration of males reduced the CAIII levels to those of the females. Treatment with testosterone for three weeks restored the copulatory behaviour in the males castrated at adult age, but restored only partially the levels of CAIII. No significant effects of the endocrine manipulations were seen on CAII. Oophorectomy, with or without testosterone substitution, had no significant effect on CAII and CAIII levels in female rats. Immunohistochemistry and histochemistry showed that the regulation of CAIII is confined to perivenous hepatocytes. CAIII can therefore serve as a useful marker in the separation of these cells. CAIII appears to belong to the proteins and enzymes of the rat liver, known to be regulated via the hypothalamo-pituitary-liver axis. It may be used as a model of gene regulation in perivenous hepatocytes.

Cataracts after autologous bone marrow transplantation in children.

We recorded the incidence and degree of posterior subcapsular cataract (PSC) in 29 children who had undergone autologous (n = 28) or syngeneic (n = 1) bone marrow transplantation (BMT) due to haematologic or lymphoid malignancy. Conditioning prior to transplantation consisted either of a combination of chemotherapy and total body irradiation (TBI) (n = 21) or of chemotherapy only (n = 8). TBI was given in one fraction of 7.5 Gy. Nine patients had received previous cranial irradiation. The patients were followed for 4-10y (median 8 y) after transplantation. Of 29 patients, 22 developed PSC, all within 4 y after BMT. With the exception of one patient who developed unilateral PSC, all had received TBI. Conversely, 100% of those who received TBI developed PSC. In this group (+TBI), eight patients (38%) developed significant PSC, defined as best corrected visual acuity <0.8 in either eye. Six patients (10 eyes) have since needed surgical repair consisting of extracapsular cataract extraction and intraocular lens implantation. There was no clear relationship between previous cranial irradiation and cataract development, nor any other obvious baseline differences between those in the +TBI group who developed significant PSC and those who did not. Although effects of previous therapy cannot be ruled out, TBI appears to be the main cause of PSC in this group of patients. Twelve patients in the +TBI group had well-preserved visual acuity throughout the study, reflecting a slow progression of PSC. This compares favourably with previous reports of allogeneic BMT, possibly owing to less need for corticosteroids after autologous BMT. We conclude that the incidence of PSC was high after autologous BMT where the conditioning regimen included total body irradiation.

Prepubertal growth and growth hormone secretion in children after treatment for hematological malignancies, including autologous bone marrow transplantation.

Prepubertal growth standards were used to assess growth in 20 children who had undergone autologous bone marrow transplantation (ABMT) as part of their treatment for hematological malignancy. Most of the patients (16 of 20) were transplanted after a relapse of their disease. A negative change in height standard deviation score (H-SDS) was seen only in the group of patients (n = 7) who had received both cranial irradiation therapy (CRT) and 7.5-Gy single-fraction total body irradiation (TBI). Height changes in this group were observed from the time of diagnosis. In contrast, the groups of patients conditioned with chemotherapy only (n = 3) or both chemotherapy and TBI, without preceding CRT (n = 10), did not demonstrate a significant loss in H-SDS. Weight related to height demonstrated large individual differences over time. Spontaneous growth hormone (GH) secretion, as measured by a four-point sleep curve, was followed longitudinally and an increasing proportion of patients with low peak levels was seen in all patient groups. In summary, prepubertal growth was suppressed only in patients who received cranial irradiation before ABMT. Despite low GH peak levels, normal prepubertal growth was found in patients with no CRT before ABMT.