Reactivation of apolipoprotein II gene transcription by cycloheximide reveals two steps in the deactivation of estrogen receptor-mediated transcription.

In this report, we describe apolipoprotein II (apoII) gene expression in cell lines derived by stable expression of the chicken estrogen receptor in LMH chicken hepatoma cells. In cell lines expressing high levels of receptor (LMH/2A), apoII gene expression is increased by estrogen 300-fold compared with levels in the receptor-deficient parent LMH line. LMH/2A cells show apoII mRNA induction and turnover kinetics similar to those in chicken liver. Inhibition of protein synthesis with cycloheximide (CHX) or puromycin following estrogen withdrawal superinduces apoII mRNA without affecting apoII mRNA stability. Superinduction is due to an estrogen-independent reactivation of apoII gene transcription. The apoII gene can be reactivated by CHX for up to 24 h following hormone withdrawal, suggesting that the gene is in a repressed yet transcriptionally competent state. These results reveal two distinct events necessary for termination of estrogen receptor-mediated transcription. The first event, removal of hormone, is sufficient to stop transcription when translation is ongoing. The second event is revealed by the CHX-induced superinduction of apoII mRNA following hormone withdrawal. This superinduction suggests that deactivation of estrogen receptor-mediated transcription requires a labile protein. Furthermore, reactivation of apoII gene expression by CHX and estrogen is additive, suggesting that estrogen is unable to overcome repression completely. Thus, a labile protein may act to repress estrogen receptor-mediated transcription of the apoII gene.

Heparin decreases the degradation rate of hepatic lipase in Fu5AH rat hepatoma cells. A model for hepatic lipase efflux from hepatocytes.

The mechanism for the stimulation of hepatic lipase secretion by heparin was studied in cultured Fu5AH rat hepatoma cells. Quantitative immunoprecipitation followed by electrophoresis and fluorography were used to isolate and quantitate the radioactive enzyme; hepatic lipase protein mass was quantitated by ELISA. Addition of heparin to the medium resulted in a 2-fold increase in lipase secretion rate, whereas cell-surface-associated and intracellular lipase decreased by 76 and 20%, respectively. Rates of synthesis of hepatic lipase measured by incorporation of Trans 35S-label into enzyme protein were not different in control or heparin-treated dishes. In pulse-chase studies, it was estimated that the degradation rate constants for control and heparin-treated cultures were 0.51 +/- 0.09 and 0.14 +/- 0.13 h-1 for control and heparin-treated cultures, respectively. 52% of the synthesized enzyme was degraded in control cultures; addition of heparin to the culture medium reduced this figure to 11% of the synthetic rate. Equilibrium binding data of highly purified 125I-hepatic lipase to Fu5AH cells at 4 degrees C demonstrate the presence of a class of high-affinity binding sites. At 37 degrees C, cell-surface-bound 125I-hepatic lipase is internalized and either degraded or recycled to the medium. The half-intracellular residence times of hepatic lipase were 55 and 31 min in control and heparin-treated cultures, respectively. Radioactivity incorporated in the 55.4 kDa high-mannose-containing lipase and the mature 57.6 kDa species was measured as a means of locating the enzyme in the secretory pathway before or beyond the medial Golgi. The disappearance of the 55.4 kDa species from the cell is similar in control and heparin-treated cultures with half-intracellular residence times of 29 and 25 min, respectively. In contrast, the amount of radiolabeled 57.6 kDa species in control cells remained constant from 15 min to 2 h, whereas it decreased by 79% in heparin-treated cells. The above data demonstrate that the increase in hepatic lipase secretion is due to a decreased degradation rate with no change in synthetic rate and that heparin primarily affected the residence time of hepatic lipase in the medial Golgi-plasma membrane region.

Isolation and characterization of clones for the rat hepatic lipase gene upstream regulatory region.

Genomic clones for 2287 nucleotides of the 5' flanking region, 135 nucleotides of the first exon, and 283 nucleotides of the first intron of the hepatic lipase gene were characterized. The predominant start site for transcription was identified by primer extension and S1 nuclease analyses to be 50 bases upstream of the ATG initiation codon. Based on the location of the major transcription start site, the functional TATA box is located 29 nucleotides upstream. Putative response elements for AP-2, cAMP, OCT-1, C/EBP, estrogen, glucocorticoids, sterols and thyroid hormone were located in this gene. Also a putative liver-specific element for apolipoproteins, C3P, was identified.

Response of children with high-risk acute lymphoblastic leukemia treated with and without cranial irradiation: a report from the Children's Cancer Group.

PURPOSE: Intensified intrathecal (i.t.) chemotherapy without cranial radiation therapy (CRT) prevents CNS relapse in children with low-risk and intermediate-risk acute lymphoblastic leukemia (ALL). In the current study, high-risk ALL patients who achieved a rapid early response (RER) to induction chemotherapy were randomized to receive intensive systemic chemotherapy and presymptomatic CNS therapy that consisted of either i.t. methotrexate (MTX) and CRT or intensified i.t. MTX alone. PATIENTS AND METHODS: Children (n = 636) with high-risk ALL (aged 1 to 9 years and WBC count > or = 50,000/microL or age > or = 10 years, excluding those with lymphomatous features) who achieved an RER (< or = 25% marrow blasts on day 7) to induction therapy and lacked CNS disease at diagnosis were randomized to receive systemic therapy with either i.t. MTX and CRT (regimen A, n = 317) or intensified i.t. MTX alone (regimen B, n = 319). RESULTS: Interim analysis in July 1993 revealed 3-year event-free survival (EFS) estimates of 82.1% +/- 4.0% (SD)and 70.4% +/- 4.2% for patients treated on regimens A and B, respectively (P = .004). As of January 1996, outcome had changed: 5-year EFS estimates were 69.1% +/- 3.4% and 75.0% +/- 2.7% for regimens A and B, respectively (P = 0.50). Marrow relapses comprised 57 events on regimen A and 43 events on regimen B. Fewer late events occurred on regimen B. CONCLUSION: For high-risk pediatric ALL patients who show an RER to induction therapy and are treated with systemic Children's Cancer Group (CCG)-modified Berlin-Frankfurt-Munster (BFM) chemotherapy, presymptomatic CNS therapy that consists of either i.t. MTX plus CRT or intensified i.t. MTX alone results in a similar 5-year EFS outcome. Furthermore, intensified i.t. MTX may protect against late bone marrow relapse.

Randomized comparison of cisplatin/vincristine/fluorouracil and cisplatin/continuous infusion doxorubicin for treatment of pediatric hepatoblastoma: A report from the Children's Cancer Group and the Pediatric Oncology Group.

PURPOSE: Previous studies demonstrated that chemotherapy with either cisplatin, vincristine, and fluorouracil (regimen A) or cisplatin and continuous infusion doxorubicin (regimen B) improved survival in children with hepatoblastoma. The current trial is a randomized comparison of these two regimens. PATIENTS AND METHODS: Patients (N = 182) were enrolled onto study between August 1989 and December 1992. After initial surgery, patients with stage I-unfavorable histology (UH; n = 43), stage II (n = 7), stage III (n = 83), and stage IV (n = 40) hepatoblastoma were randomized to receive regimen A (n = 92) or regimen B (n = 81). Patients with stage I-favorable histology (FH; n = 9) were treated with four cycles of doxorubicin alone. RESULTS: There were no events among patients with stage I-FH disease. Five-year event-free survival (EFS) estimates were 57% (SD = 5%) and 69% (SD = 5%) for patients on regimens A and B, respectively (P =.09) with a relative risk of 1.54 (95% confidence interval, 0.93 to 2.5) for regimen A versus B. Toxicities were more frequent on regimen B. Patients with stage I-UH, stage II, stage III, or stage IV disease had 5-year EFS estimates of 91% (SD = 4%), 100%, 64% (SD = 5%), and 25% (SD = 7%), respectively. Outcome was similar for either regimen within disease stages. At postinduction surgery I, patients with stage III or IV disease who were found to be tumor-free had no events; those who had complete resections achieved a 5-year EFS of 83% (SD = 6%); other patients with stage III or IV disease had worse outcome. CONCLUSION: Treatment outcome was not significantly different between regimen A and regimen B. Excellent outcome was achieved for patients with stage I-UH and stage II hepatoblastoma and for subsets of patients with stage III disease. New treatment strategies are needed for the majority of patients with advanced-stage hepatoblastoma.

Clinical features and treatment outcome of childhood T-lineage acute lymphoblastic leukemia according to the apparent maturational stage of T-lineage leukemic blasts: a Children's Cancer Group study.

PURPOSE: Leukemic cells from T-lineage acute lymphoblastic leukemia (ALL) patients are thought to originate from T-lymphocyte precursors corresponding to discrete stages of T-cell ontogeny. Here we sought to determine the influence of leukemic cell apparent maturational stage on treatment outcomes in pediatric T-lineage ALL. PATIENTS AND METHODS: From 1983 through 1993, 407 pediatric T-lineage ALL patients were enrolled onto two sequential series of risk-adjusted treatment protocols of the Children's Cancer Group. In the current analysis, T-lineage ALL patients were immunophenotypically classified as follows: CD7+ CD2- CD5- pro-thymocyte leukemia (pro-TL), CD7+ (CD2 or CD5)+ CD3- immature TL, and CD7+ CD2+ CD5+ CD3+ mature TL. RESULTS: Similar induction outcomes of 91.4%, 97.1%, and 98.3% were obtained by the pro-, immature, and mature TL groups, respectively. Four-year event-free survival (EFS) was lower for pro-TL patients (57.1%; SD = 8.4%,) compared with immature and mature TL patients (68.5%; SD = 3.5%; and 77.1%; SD = 4.0%, respectively) with an overall significance of .05 (log-rank test) or .04 (log-rank trend test). Relative hazards rates (RHR) were 2.11 and 1.22 for pro-TL and immature TL versus mature TL, respectively. Highly significant differences were found for overall survival (P = .005, log-rank test; P = .009, log-rank trend test). Multivariate analysis confirmed that the prognostic influence of ontogeny grouping was independent of that of other prognostic factors. CONCLUSION: Leukemic cells of the pro-TL maturation stage identify a small subgroup of T-lineage ALL patients who have a significantly worse EFS outcome than patients whose cells are of a more mature stage of development.

Prognostic impact of trisomies of chromosomes 10, 17, and 5 among children with acute lymphoblastic leukemia and high hyperdiploidy (> 50 chromosomes).

PURPOSE: Children with acute lymphoblastic leukemia (ALL) and high hyperdiploidy (> 50 chromosomes) have improved outcome compared with other ALL patients. We sought to identify cytogenetic features that would predict differences in outcome within this low-risk subset of ALL patients. MATERIALS AND METHODS: High-hyperdiploid ALL patients (N = 480) were enrolled between 1988 and 1995 on Children's Cancer Group (CCG) trials. Karyotypes were determined by conventional banding. Treatment outcome was analyzed by life-table methods. RESULTS: Patients with 54 to 58 chromosomes had better outcome than patients with 51 to 53 or 59 to 68 chromosomes (P = .0002). Patients with a trisomy of chromosome 10 (P<.0001), chromosome 17 (P = .0002), or chromosome 18 (P = .004) had significantly improved outcome compared with their counterparts who lacked the given trisomy. Patients with a trisomy of chromosome 5 had worse outcome than patients lacking this trisomy (P = .02). Patients with trisomies of both chromosomes 10 and 17 had better outcome than those with a trisomy of chromosome 10 (P = .09), a trisomy of chromosome 17 (P =.01), or neither trisomy (P<.0001). Multivariate analysis indicated that trisomy of chromosome 10 (P = .001) was the most significant prognostic factor for high-hyperdiploid patients, yet trisomy of chromosome 17 (P =.02) or chromosome 5 (P = .01) and modal chromosome number (P = .02) also had significant multivariate effects. CONCLUSION: Trisomy of chromosomes 10 and 17 as well as modal chromosome number 54 to 58 identify subgroups of patients with high-hyperdiploid ALL who have a better outcome than high-hyperdiploid patients who lack these cytogenetic features. Trisomy of chromosome 5 confers poorer outcome among high-hyperdiploid patients.

Clinical significance of translocation t(1;19) in childhood acute lymphoblastic leukemia in the context of contemporary therapies: a report from the Children's Cancer Group.

PURPOSE: The nonrandom translocation t(1;19) has been associated with poor outcome in pediatric B-lineage acute lymphoblastic leukemia (ALL). Because most patients treated by contemporary therapies now achieve improved outcomes, we have reassessed the prognostic significance of t(1;19). PATIENTS AND METHODS: Cytogenetic data were accepted for 1,322 children (<21 years old) with newly diagnosed ALL enrolled between 1988 and 1994 on risk-adjusted studies of the Children's Cancer Group (CCG). Forty-seven patients (3.6%) were t(1;19) positive (+); 1,275 (96.4%) were t(1;19) negative (-). Clinical characteristics and treatment outcome were compared using standard methods. RESULTS: Translocation (1;19)+ patients were more likely than t(1;19)- patients to be 10 years of age or greater (P < .001) or CD10+ CD19+ CD34- (P < .0001), or nonwhite (P = .02). Patients with a balanced t(1;19) were less likely to be hyperdiploid than patients with an unbalanced der(19)t(1;19). Event-free survival (EFS) was similar for the overall group of t(1;19)+ and t(1;19)- patients, with 4-year estimates of 69.5% (SD, 6.8%) and 74.8% (SD, 1.3%; P = .48), respectively. However, patients with unbalanced der(19)t(1;19) had significantly better outcomes than patients with balanced t(1;19): 4-year EFS were 80.6% (SD, 7.1%) and 41.7% (SD, 13.5%), respectively (P = .003). These differences were maintained within the individual studies analyses and after exclusion of t(1;19)+ patients whose cells were hyperdiploid with more than 50 chromosomes. CONCLUSION: The overall group of t(1;19)+ patients, as well as the subgroup with an unbalanced der(19)+ (1;19) had outcomes similar to that of t(1;19)- patients, whereas patients with balanced t(1;19) had poorer outcomes. Thus, although the overall prognostic significance of t(1;19) has been obviated by contemporary risk-adjusted protocols, the balanced t(1;19) translocation remains an adverse prognostic factor.

Duration of hospitalization as a measure of cost on Children's Cancer Group acute lymphoblastic leukemia studies.

PURPOSE: We used duration of hospitalization as a surrogate for cost and event-free survival as a measure of effectiveness to estimate the cost-effectiveness ratios of various treatment regimens on Children's Cancer Group trials for acute lymphoblastic leukemia. PATIENTS AND METHODS: The analyses included 4,986 children (2 to 21 years of age) with newly diagnosed acute lymphoblastic leukemia enrolled onto risk-adjusted protocols between 1988 and 1995. Analyses were based on a model of 100 patients. The marginal cost-effectiveness ratio (hospital days per additional patient surviving event-free) was the difference in total duration of hospitalization divided by the difference in number of event-free survivors at 5 years for two regimens. Relapse-adjusted marginal cost of frontline therapy was the difference in total duration of hospitalization for frontline therapy plus relapse therapy divided by the difference in number of event-free survivors at 5 years on the frontline therapy for two regimens. RESULTS: One or two delayed intensification (DI) phases, augmented therapy, and dexamethasone all improved outcome. Marginal cost-effectiveness of these regimens compared with the control regimens was 133 days per patient for DI, 117 days per patient for double DI, and 41 days per patient for augmented therapy. Dexamethasone resulted in 17 fewer days per patient. Relapse-adjusted marginal costs were 68 days per patient for DI and 52 days for double DI. Augmented therapy and dexamethasone-based therapy resulted in 16 and 82 fewer hospital days, respectively. The estimated cost-effectiveness for treating any first relapse was 250 days per patient. CONCLUSION: DI, double DI, augmented therapy, and dexamethasone-based therapy are cost-effective strategies compared with current treatment of first relapse.

Frequency and clinical significance of cytogenetic abnormalities in pediatric T-lineage acute lymphoblastic leukemia: a report from the Children's Cancer Group.

PURPOSE: Nonrandom chromosomal translocations are frequently observed in pediatric patients with acute lymphoblastic leukemia (ALL). Specific translocations, such as t(4;11) and t(9;22), identify subgroups of B-lineage ALL patients who have an increased risk of treatment failure. The current study was conducted to determine the prognostic significance of chromosomal translocations in T-lineage ALL patients. MATERIALS AND METHODS: The study included 169 children with newly diagnosed T-lineage ALL enrolled between 1988 and 1995 on risk-adjusted protocols of the Children's Cancer Group (CCG) who had centrally reviewed cytogenetics data. Outcome analyses used standard life-table methods. RESULTS: Presenting features for the current cohort were similar to those of concurrently enrolled patients for whom cytogenetic data were not accepted on central review. The majority of patients (80.5%) were assigned to CCG protocols for high-risk ALL and 86.4% had pseudodiploid (n = 80) or normal diploid (n = 66) karyotypes; modal chromosome number was not a significant prognostic factor. Overall, 103 of 169 (61%) patients had an abnormal karyotype, including 31 with del(6q), 29 with 14q11 breakpoints, 15 with del(9p), 11 with trisomy 8, nine with 11q23 breakpoints, nine with 14q32 translocations, and eight with 7q32-q36 breakpoints. Thirteen patients had the specific 14q11 translocation t(11;14)(p13;q11) and all were classified as poor risk. Patients with any of these translocations had outcomes similar to those with normal diploid karyotypes. CONCLUSION: Chromosomal abnormalities, including specific nonrandom translocations, were frequently observed in a large group of children with T-lineage ALL, but were not significant prognostic factors for this cohort. Thus, contemporary intensive treatment programs result in favorable outcomes for the majority of T-lineage ALL patients, regardless of karyotypic abnormalities, and such features do not identify patients at higher risk for relapse.

Expression of aberrantly spliced oncogenic ikaros isoforms in childhood acute lymphoblastic leukemia.

PURPOSE: We sought to determine if molecular abnormalities involving the Ikaros gene could contribute to the development of acute lymphoblastic leukemia (ALL) in children. PATIENTS AND METHODS: We studied Ikaros gene expression in normal human bone marrow, normal thymocytes, normal fetal liver-derived immature lymphocyte precursor cell lines, eight different ALL cell lines, and leukemic cells from 69 children with ALL (T-lineage ALL, n = 18; B-lineage ALL, n = 51). Expression of Ikaros protein and its subcellular localization were examined by immunoblotting and confocal laser-scanning microscopy, respectively. Polymerase chain reaction (PCR) and nucleotide sequencing were used to identify the specific Ikaros isoforms expressed in these cells. Genomic sequencing of splice junction regions of the Ikaros gene was performed in search for mutations. RESULTS: In each of the ALL cases, we found high-level expression of a non-DNA-binding or aberrant DNA-binding isoform of Ikaros with abnormal subcellular compartmentalization patterns. In contrast, only wild-type Ik-1 and Ik-2 isoforms with normal subcellular localization were found in normal bone marrow cells and thymus-derived or fetal liver-derived normal lymphocyte precursors. In leukemic cells expressing the aberrant Ikaros coding sequences with the 30-base-pair deletion, genomic sequence analysis of the intron-exon junctions between exons 6 and 7 yielded the wild-type sequence. We identified a single nucleotide polymorphism (SNP) affecting the third base of the triplet codon for a proline (CCC or CCA) in the highly conserved bipartite activation region (viz, A or C at position 1002 numbering from the translation start site of Ik-1) within our Ikaros clones. Bi-allelic expression of truncated and/or non-DNA-binding isoforms along with wild-type isoforms was observed in leukemic cells, which implicates trans-acting factor(s) affecting splice site recognition. CONCLUSION: Our findings link specific molecular defects involving the Ikaros gene to childhood ALL. Posttranscriptional regulation of alternative splicing of Ikaros RNA seems to be defective in leukemic lymphocyte precursors from most children with ALL. Consequently, leukemic cells from ALL patients, in contrast to normal lymphocyte precursors, express high levels of non-DNA-binding Ikaros isoforms that are reminiscent of the non-DNA-binding Ikaros isoforms that lead to lymphoblastic leukemia in mice.

Abnormalities of chromosome bands 13q12 to 13q14 in childhood acute lymphoblastic leukemia.

PURPOSE: Little is known about nonrandom deletions of chromosome bands 13q12 to 13q14 (13q12-14) in acute lymphoblastic leukemia (ALL). We determined the prognostic significance of cytogenetically identified breakpoints in 13q12-14 in children with newly diagnosed ALL treated on Children's Cancer Group protocols from 1988 to 1995. PATIENTS AND METHODS: Breakpoints in 13q12-14 were identified in 36 (2%) of the 1,946 cases with accepted cytogenetic data. Outcome analysis used standard life-table methods. RESULTS: Seventeen patients (47%) with an abnormal 13q12-14 were classified, according to the National Cancer Institute (NCI), as poor risk, and 15 patients (42%) were standard risk; four (11%) were infants less than 12 months of age. Eight cases had balanced rearrangements of 13q12-14, 27 patients had a partial loss of 13q, and one had both a partial gain and a partial loss. The most frequent additional abnormalities among these patients were an abnormal 12p, a del(6q), a del(9p), a 14q11 breakpoint, and an 11q23 breakpoint. Nineteen patients were pseudodiploid, 10 were hyperdiploid, and seven were hypodiploid. Patients with an abnormal 13q12-14 had significantly worse event-free survival than patients lacking such an abnormality, with estimates at 6 years of 61% (SD = 14%) and 74% (SD = 1%), respectively (P =.04; relative risk = 1.74). Overall survival, however, was similar for the two groups (P =.25). The prognostic effect of an abnormal 13q was attenuated in a multivariate analysis adjusted for NCI risk status and ploidy (P =.72). CONCLUSION: Aberrations of 13q12-14 may contribute to leukemogenesis of childhood ALL and confer increased risk of treatment failure but are associated with other poor-risk features.

Treatment outcome and prognostic factors for infants with acute lymphoblastic leukemia treated on two consecutive trials of the Children's Cancer Group.

PURPOSE: Infants represent a very poor risk group for acute lymphoblastic leukemia (ALL). We report treatment outcome for such patients treated with intensive therapy on consecutive Children's Cancer Group (CCG) protocols. PATIENTS AND METHODS: Between 1984 and 1993, infants with newly diagnosed ALL were enrolled onto CCG-107 (n = 99) and CCG-1883 (n = 135) protocols. Postconsolidation therapy was more intensive on CCG-1883. On both studies, prophylactic treatment of the CNS included both high-dose systemic chemotherapy and intrathecal therapy, in contrast to whole-brain radiotherapy, which was used in earlier studies. RESULTS: Most patients (>95%) achieved remission with induction therapy. The most frequent event was a marrow relapse (46 patients on CCG-107 and 66 patients on CCG- 1883). Four-year event-free survival was 33% (SE = 4.7%) on CCG-107 and 39% (SE = 4.2%) on CCG- 1883. Both studies represent an improvement compared with a 22% (SE = 5.1%) event-free survival for historical controls. Four-year cumulative probabilities of any marrow relapse or an isolated CNS relapse were, respectively, 49% (SE = 5%) and 9% (SE = 3%) on CCG-107 and 50% (SE = 5%) and 3% (SE = 2%) on CCG-1883, compared with 63% (SE = 6%) and 5% (SE = 3%) for the historical controls. Independent adverse prognostic factors were age less than 3 months, WBC count of more than 50,000/microL, CD10 negativity, slow response to induction therapy, and presence of the translocation t(4;11). CONCLUSION: Outcome for infants on CCG-107 and CCG- 1883 improved, compared with historical controls. Marrow relapse remains the primary mode of failure. Isolated CNS relapse rates are low, indicating that intrathecal chemotherapy combined with very-high-dose systemic therapy provides adequate protection of the CNS. The overall unsatisfactory outcome observed for the infant ALL population warrants the future use of novel alternative therapies.

Children's Cancer Group trials in childhood acute lymphoblastic leukemia: 1983-1995.

Since 1968, the Children's Cancer Group (CCG) has treated more than 16,000 children with acute lymphoblastic leukemia (ALL). Herein, we report improvements obtained in CCG trials during two successive series of studies (1983-1988 and 1989-1995). Overall, 10-year EFS was 62% +/- 10% for the 1983-1988 series and 72% +/- 1% for the 1988-1995 series (P< 0.0001). Five-year cumulative rates of isolated CNS relapses were 5.9% and 4.4%. Therapy based on the Berlin-Frankfurt-Münster 76/79 study improved outcomes for intermediate and higher risk patients in the first series. For intermediate risk patients, delayed intensification (DI) was most crucial for improved outcome and cranial irradiation was safely replaced with maintenance intrathecal methotrexate, providing patients received intensified systemic therapy. In the second series, randomized trials showed better outcome with one vs no DI phase for lower risk patients, with two vs one DI phase for intermediate risk patients, and with the CCG 'augmented regimen' for higher risk patients with a slow day 7 marrow response. Cranial irradiation was safely replaced with additional intrathecal methotrexate for higher risk patients with a rapid day 7 marrow response. In a subsequent study, substitution of dexamethasone in place of prednisone in induction and maintenance improved outcome for standard risk patients. All patients received dexamethasone in DI. These successful treatment strategies form the basis for our current ALL trials.

Residual bone marrow leukemic progenitor cell burden after induction chemotherapy in pediatric patients with acute lymphoblastic leukemia.

We used highly sensitive multiparameter flow cytometry and blast colony assays to quantify the leukemic progenitor cell (LPC) burden of postinduction chemotherapy bone marrows from newly diagnosed and relapsed pediatric patients with acute lymphoblastic leukemia (ALL). Of 890 newly diagnosed patients, 243 (27%) had detectable LPC in the postinduction bone marrow samples with an average (mean +/- SE) LPC content of 22+/-9 LPC/10(6) mononuclear cell (MNC; range, 0-7199/10(6) MNC; median, 0/10(6) MNC). By comparison, 24 of 50 (48%) patients with relapsed ALL had detectable LPC in their postinduction bone marrow specimens (P = 0.003), and their average LPC content was 202+/-139 LPC/10(6) MNC. Fewer patients with B-lineage ALL (170 of 786; 22%) than patients with T-lineage ALL (73 of 104; 70%) harbored residual LPC in their postinduction bone marrow specimens (P < 0.0001). This correlation with immunophenotype was independent of the National Cancer Institute risk classification. Similarly, 19 of 44 (43%) patients with relapsed B-lineage ALL versus 5 of 6 (83%) patients with relapsed T-lineage ALL harbored residual LPC in their postinduction bone marrow specimens (P = 0.09). Among newly diagnosed patients, those with high-risk ALL seemed to have larger numbers of residual LPC in their bone marrow after induction chemotherapy than those with standard risk ALL (53+/-26, n = 286 versus 7+/-1, n = 604, P = 0.04). LPC of patients with standard risk ALL who had a slow early marrow response at day 7 seemed to be more resistant to the three-drug induction chemotherapy than patients who had a rapid early marrow response. Overall, the order of chemosensitivity of LPC was: newly diagnosed standard risk B-lineage > newly diagnosed higher risk B-lineage > newly diagnosed standard risk T-lineage > newly diagnosed higher risk T-lineage > relapsed B-lineage > relapsed T-lineage. Notably, LPC- patients whose end-of-induction remission bone marrow specimens had zero LPC had an excellent early event-free survival outcome. Within the standard and high-risk subsets, LPC- patients had a 2.6-fold lower and 2.4-fold lower incidence of events, respectively, than LPC+ patients. At 6 months, 12 months, as well as 24 months, the ranking order for better event-free survival was: standard risk, LPC- > high risk, LPC- > standard risk, LPC+ > high risk, and LPC+.

Paucity of leukemic progenitor cells in the bone marrow of pediatric B-lineage acute lymphoblastic leukemia patients with an isolated extramedullary first relapse.

Isolated extramedullary relapse in childhood acute lymphoblastic leukemia (ALL) may be accompanied by occult bone marrow disease. We used a highly sensitive assay to quantify leukemic progenitor cells (LPCs) in the bone marrow of such patients. Multiparameter flow cytometry and blast colony assays were used to detect LPCs in the bone marrow of 31 pediatric B-lineage ALL patients with an isolated extramedullary first relapse. Sites of relapse were central nervous system (22 patients), testes (7 patients), and eye (2 patients). Bone marrow (BM) LPC counts ranged from 0/10(6) mononuclear cells (MNCs) to 356/10(6) MNCs (mean +/- SE, 27.8+/-13.1/10(6) MNCs). LPCs were undetectable in 19 patients (61%). The BM LPC burden at the time of extramedullary relapse was similar, regardless of site (Wilcoxon P = 0.77) or time of relapse (Wilcoxon P = 0.80). Compared with higher risk, standard risk at initial diagnosis showed a trend for increased BM LPC burden (mean +/- SE, 44.6+/-17.1 versus 7.5+/-3.3; Wilcoxon P = 0.22). After successful postrelapse induction chemotherapy, LPC counts in 21 evaluated patients ranged from 0/10(6) to 175/10(6) MNCs (mean +/- SE, 15.9+/-9.6/10(6) MNCs). By comparison, LPC burden was higher after successful induction chemotherapy among children with an early BM relapse (range, 0 to 3262/ 106 MNC; mean +/- SE, 166+/-107; Wilcoxon P = 0.11). Thus, not all patients with an extramedullary relapse have occult systemic failure with substantial involvement of the bone marrow, and after reinduction therapy, LPC counts were lower in these patients than in patients treated for an overt BM first relapse.

Expression of dominant-negative Ikaros isoforms in T-cell acute lymphoblastic leukemia.

Ikaros, a zinc finger-containing DNA-binding protein, is required for normal lymphocyte development. Germ-line mutant mice that express only non-DNA binding dominant-negative "leukemogenic" Ikaros isoforms lacking critical NH2-terminal zinc fingers develop an aggressive form of T-cell leukemia. We studied Ikaros gene expression in leukemic cells from 18 children with T-cell acute lymphoblastic leukemia (T-ALL). In each of the 18 T-ALL cases as well as JK-E6-1 and MOLT-3 cell lines, we found high-level expression of dominant-negative isoforms of Ikaros with abnormal subcellular compartmentalization patterns. Nuclear extracts from these cells failed to bind to the IKAROS-specific binding sequence in DNA. PCR cloning and sequencing confirmed that JK-E6-1 and MOLT-3 cell lines as well as leukemic cells from 9 of 10 patients with T-ALL expressed dominant-negative Ikaros isoforms Ik-4, Ik-7, and Ik-8 that lack critical NH2-terminal zinc fingers. In 6 of 10 patients, we detected a specific mutation leading to an in-frame deletion of 10 amino acids (delta KSSMPQKFLG) upstream to the transcription activation domain and adjacent to the COOH-terminal zinc fingers of Ik-2, Ik-4, Ik-7, and Ik-8. Thus, children with T-ALL express high levels of dysfunctional dominant-negative Ikaros isoforms.

Amino acid differences in the N-terminus of C(H)2 influence the relative abilities of IgG2 and IgG3 to activate complement.

The four human IgG isotypes are highly conserved in amino acid sequence, but show differential ability to activate complement (C'): IgG3 and IgG1 are very active, IgG2 is active under certain conditions, and IgG4 is inactive. Although the second constant domain [C(H)2] is critical for C' activation, the individual amino acids that confer isotype-specific activity have not been identified. We have generated a series of mutants between IgG2 and IgG3, resulting in the exchange of the four N-terminal and six C-terminal polymorphic residues within C(H)2. Mutants containing the N-terminus of the C(H)2 of IgG3 were as effective as wildtype IgG3 in C1q binding, C1 activation and terminal complex (MAC) formation, but had reduced ability to effect C'-mediated lysis. IgG2 and mutants containing the N-terminal portion of the C(H)2 of IgG2 were reduced compared to IgG3 in activating C1, binding C1q and inducing assembly of the MAC, and were inactive in mediating lysis of target cells. Thus, the amino acid sequence differences in the N-terminus of C(H)2 play a critical role in determining the relative abilities of IgG2 and IgG3 to bind C1q and activate the C' cascade although additional residues of C(H)2 must be involved in mediating optimal target cells lysis. The sequence of the N-terminus of C(H)2 was less critical in determining C4 and C3 binding. Characterization of domain exchange mutants suggests that intermediate steps may be partly dependent on domains other than C(H)2. IgGs that do not direct target cell lysis nevertheless activate intermediate steps in the pathway, which may contribute to immune complex-associated disorders.

Hemophilia therapy innovation: development of an advanced category recombinant factor VIII by a plasma/albumin-free method. Proceedings of a Special Symposium at the XIXth Congress of the International Society on Thrombosis and Haemostasis, July 12-18, 2003, Birmingham, UK.

Replacement therapy for hemophilia A has evolved from the early use of whole blood, citrated plasma, and cryoprecipitate, to purified factor VIII (FVIII) concentrates, first derived from plasma, then produced by recombinant DNA technology. Recombinant FVIII (rFVIII) concentrates have provided improved safety for patients with hemophilia A since they significantly reduce the risk of transmission of blood-borne infections. Nevertheless, human- or animal-derived plasma proteins are still included at some step in preparation of all previously licensed rFVIII, thereby introducing the potential for transmission of human or animal pathogens. Anti-hemophilic factor (recombinant), plasma/albumin-free method (rAHF-PFM), a novel advanced category rFVIII produced without the addition of human or animal plasma proteins, has been developed with the goal of providing the greatest possible margin of safety to hemophilia patients. This report, based on a symposium of the XIXth International Society on Thrombosis and Haemostasis Congress, provides an overview of the rAHF-PFM development program as well as current findings from the global clinical evaluation of rAHF-PFM in pediatric and adult previously treated patients.

Prognostic significance of the CD10+CD19+CD34+ B-progenitor immunophenotype in children with acute lymphoblastic leukemia: a report from the Children's Cancer Group.

Leukemic cells from most patients with B-lineage acute lymphoblastic leukemia (ALL) appear to originate from normal B-lymphocyte precursors. The earliest B-cell progenitors coexpress the antigens CD10, CD19, or CD34 on their cell surfaces. In a large cohort of 2028 children with ALL, we compared treatment outcomes of a subset of B-lineage ALL patients with CD10+CD19+CD34+ immature B-progenitor leukemia (BPL) to the treatment outcomes of the remaining CD19+ B-lineage ALL patients. Pediatric B-lineage ALL cases enrolled on risk-adjusted ALL treatment protocols of the Children's Cancer Group were immunophenotypically classified as BPL or non-BPL. Patients were stratified further into age groups of > or = 1 year and <12 months. Event-free survival (EFS) outcomes were calculated by standard life table methods. BPL patients in both age groups generally had more favorable presenting characteristics than non-BPL controls. Within the age group of > or = 1 year, BPL patients had a slightly better EFS outcome than non-BPL patients, with 3-year estimates of 83.9% (SD = 1.1%) vs. 78.8% (SD = 1.8%), respectively (P = 0.10). Infants with BPL, representing one-fifth of the total infant patient population, had a significantly better EFS outcome than infants with non-BPL (three-year EFS: 82.4%, SD = 9.2% vs. 34.4%, SD = 5.9%, P = 0.006). In univariate analyses, the relative hazard rate (RHR) was 3.73 for non-BPL vs BPL and this marked difference in EFS outcome was maintained at 5 years of follow-up. The favorable prognostic influence of the BPL immunophenotype for infants remained significant in multivariate analyses with an RHR of 2.72 for non-BPL vs BPL (P = 0.05). CD10+CD19+CD34+ immature B-progenitor immunophenotype is associated with favorable characteristics for children with ALL and identifies a subset of infants who achieve favorable EFS outcomes.