Use of a novel colony assay to evaluate the cytotoxicity of an immunotoxin containing pokeweed antiviral protein against blast progenitor cells freshly obtained from patients with common B-lineage acute lymphoblastic leukemia.

We report a novel colony assay for B-lineage progenitor cells in acute lymphoblastic leukemia (ALL). The primary plating efficiency of blast progenitors freshly obtained from common B-lineage ALL patients varied between 0.09 and 2.63%. Morphological, cytochemical, and immunological analyses of cells from day 7 colonies provided the evidence that they are B-lineage lymphoblasts. Immunological marker analyses of cultured blasts using BA-2 (anti-CD9), BA-3 (anti-CD10), BA-1 (anti-CD24), and B43 mAb have allowed us to define two distinct immunological groups. The first group had BA-2+, BA-3+, BA-1+, B43+ marker profiles, consistent with the phenotype of uncultured bone marrow blasts. The second group differed in that the cells in the blast colonies were BA-3 (anti-CD10)-negative, although many of the cells in the bulk population were BA-3+ before culture. Blasts from both groups were able to proliferate and form secondary colonies when recultured. A pan-B immunotoxin was synthesized by linking B43, a human B cell-specific mAb, to pokeweed antiviral protein (PAP). This study showed that B43-PAP can effectively eradicate leukemic progenitor cells freshly obtained from patients with common B-lineage ALL. B43-PAP eliminated greater than 99.96% of blast progenitors under conditions in which only minimal inhibition of normal bone marrow progenitor cells (CFU-GM, CFU-E, CFU-MK, CFU-GEMM) was observed. Our results establish that the surface determinant recognized by B43 is expressed on B-lineage progenitor cells in ALL, and that these cells are sensitive to PAP at the ribosomal level. To our knowledge, B43-PAP is the first IT to prove effective against common B-lineage ALL cells.

BTK as a mediator of radiation-induced apoptosis in DT-40 lymphoma B cells.

Bruton's tyrosine kinase (BTK) is a member of the SRC-related TEC family of protein tyrosine kinases (PTKs). DT-40 lymphoma B cells, rendered BTK-deficient through targeted disruption of the btk gene by homologous recombination knockout, did not undergo radiation-induced apoptosis, but cells with disrupted lyn or syk genes did. Introduction of the wild-type, or a SRC homology 2 domain or a plecstrin homology domain mutant (but not a kinase domain mutant), human btk gene into BTK-deficient cells restored the apoptotic response to radiation. Thus, BTK is the PTK responsible for triggering radiation-induced apoptosis of lymphoma B cells, and its kinase domain is indispensable for the apoptotic response.

Biotherapy of B-cell precursor leukemia by targeting genistein to CD19-associated tyrosine kinases.

B-cell precursor (BCP) leukemia is the most common form of childhood cancer and the second most common form of acute leukemia in adults. Human BCP leukemia was treated in a severe combined immunodeficient mouse model by targeting of the tyrosine kinase inhibitor Genistein (Gen) to the B cell-specific receptor CD19 with the monoclonal antibody B43. The B43-Gen immunoconjugate bound with high affinity to BCP leukemia cells, selectively inhibited CD19-associated tyrosine kinases, and triggered rapid apoptotic cell death. At less than one-tenth the maximum tolerated dose more than 99.999 percent of human BCP leukemia cells were killed, which led to 100 percent long-term event-free survival from an otherwise invariably fatal leukemia. The B43-Gen immuno-conjugate might be useful in eliminating leukemia cells in patients who have failed conventional therapy.

Detection and characterization of human high molecular weight B cell growth factor receptors on leukemic B cells in chronic lymphocytic leukemia.

Human high molecular weight-B cell growth factor (HMW-BCGF) (60 kD) stimulates activated normal B cells, B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, hairy cell leukemia (HCL) cells, prolymphocytic leukemia (PLL) cells, and chronic lymphocytic leukemia (CLL) cells. The expression of human high molecular weight B cell growth factor (HMW-BCGF) receptors (R) on clonal populations of leukemic B cells in CLL was studied by ligand binding assays using 125I-labeled HMW-BCGF as well as by immunofluorescence/flow cytometry and Scatchard analyses using an anti-HMW-BCGF R monoclonal antibody (MAb), designated BA-5. There was a high correlation between HMW-BCGF R expression and responsiveness to HMW-BCGF. 60% of CLL cases constitutively expressed HMW-BCGF R and showed a marked proliferative response to HMW-BCGF in [3H]TdR incorporation assays as well as colony assays. Similarly, HCL cells, PLL cells, and activated normal B cells expressed functional HMW-BCGF R, as determined by ligand binding assays using 125I-HMW-BCGF, [3H]TdR incorporation assays, and reactivity with BA-5 MAb. Scatchard analyses indicated the existence of approximately 3,000 HMW-BCGF R/cell on HMW-BCGF responsive CLL cells with an apparent Ka value of 4.6 X 10(7) M-1. The concentrations of HMW-BCGF required for maximum stimulation of CLL cells were two to three orders of magnitude lower than those needed for half maximal receptor occupancy, indicating that only a small fraction of HMW-BCGF R need to be occupied to stimulate leukemic CLL B cells. Crosslinking of surface bound 125I-HMW-BCGF (60 kD) with the bivalent crosslinker DTSSP to its binding site on fresh CLL cells identified a 150-kD HMW-BCGF/HMW-BCGF R complex, suggesting an apparent molecular weight of 90 kD for the receptor protein. The growth stimulatory effects of HMW-BCGF on clonogenic CLL cells did not depend on accessory cells or costimulant factors. The anti-HMW-BCGF R monoclonal antibody BA-5 disrupted HMW-BCGF/HMW-BCGF R interactions at the level of clonogenic CLL cells and inhibited HMW-BCGF-stimulated CLL colony formation in vitro. To our knowledge, this study represents the first detailed analysis of expression, function, and structure of HMW-BCGF R on B lineage CLL cells.

Intrinsic radiation resistance of primary clonogenic blasts from children with newly diagnosed B-cell precursor acute lymphoblastic leukemia.

The radiation sensitivity of primary clonogenic blasts from 44 children with newly diagnosed B-cell precursor acute lymphoblastic leukemia (ALL) was analyzed using leukemic progenitor cell (LPC) colony assays. The derived values for SF2 (surviving fraction at 200 cGy) and alpha (initial slope of radiation survival curves constructed according to the linear quadratic model) indicated a marked interpatient heterogeneity in intrinsic radiation sensitivity of LPC populations. The SF2 values ranged from 0.01 to 1.00 (median = 0.430; mean +/- SE = 0.47 +/- 0.04), and the alpha values ranged from 0.000 to 3.272 Gy-1 (median = 0.280 Gy-1; mean +/- SE = 0.430 +/- 0.093 Gy-1). When CD19+ CD34+ versus CD19+ CD34- immunophenotypes were compared, a trend toward higher SF2 and lower alpha values were observed in LPC from CD34+ patients, consistent with greater radiation resistance. When patients were divided into three approximately equal groups based on increasing levels of CD34 expression, a clear ordering effect was observed indicating that increased CD34 expression levels are associated with significantly higher radiation resistance at the level of B-lineage LPC. The highest CD34 expression group (> or = 75% positivity) had 1.4-fold higher SF2 (P = 0.05) and twofold lower alpha values (P = 0.06) than the lowest group (< 30% positivity). Furthermore, the CD34 positivity of radiation resistant (alpha < or = 0.2 and SF2 > or = 0.5) B-cell precursor ALL cases was greater than the CD34 positivity of radiation sensitive (alpha > 0.2 and/or SF2 < 0.5) cases (56 +/- 9% versus 34 +/- 9%, P = 0.09). Whereas only 6 of 16 (38%) of radiation sensitive cases were CD34+, 11 of 15 (73%) of radiation resistant cases expressed CD34 (P = 0.04). Our results offer new insights into the inherent and/or acquired radiation resistance of primary clonogenic blasts from B-cell precursor ALL patients.

Heterogeneity of cultured leukemic lymphoid progenitor cells from B cell precursor acute lymphoblastic leukemia (ALL) patients.

Colony assays were performed for 50 patients with B cell precursor acute lymphoblastic leukemia (ALL). Blast colony formation was observed for 33 patients, and the plating efficiency (PE) showed a marked interpatient variation, which indicates a pronounced biological heterogeneity at the level of leukemic progenitor cells. Notably, the mean PE of leukemic B cell precursors from patients with a pseudodiploid or near-diploid karyotype with structural chromosomal abnormalities (SCA) was significantly higher than the mean PE of normal diploid or hyperdiploid cases. All patients who had SCA involving 7p13, 11q23-24, or 12p11-13, and patients with a Philadelphia chromosome had high PE values. The S phase percentage, expression of CD19 antigen, and relapse status were also correlated with PE. Significantly, colony blasts had slightly different surface marker profiles in each case and were common ALL antigen negative in 33% of cases, which indicates the existence of a marked immunological heterogeneity at the level of leukemic progenitor cells.

Ultraviolet radiation rapidly induces tyrosine phosphorylation and calcium signaling in lymphocytes.

UV radiation is known to induce lymphocyte nonresponsiveness both in vitro and in vivo. We have found that UV radiation rapidly induced tyrosine phosphorylation and calcium signaling in normal human peripheral blood lymphocytes. In the leukemic T cell line Jurkat and the Burkitt's lymphoma cell line Ramos, UV rapidly induced tyrosine phosphorylation in a wavelength-dependent manner, giving strong signals after UVB and UVC, but not UVA, irradiation. Similarly, in Jurkat cells UV-induced calcium signals were dependent on the dose of UVB or UVC irradiation over a range of 150-1200 J/m2, but only a small signal was observed for UVA at a dose of 1200 J/m2. The UV-induced calcium signals were blocked by the tyrosine kinase inhibitor herbimycin A, indicating that they were dependent on tyrosine phosphorylation. Phospholipase C (PLC) gamma 1 was tyrosine phosphorylated in response to UV irradiation but to a lesser extent than observed after CD3 cross-linking. However, PLC gamma 1-associated proteins demonstrated to bind to the PLC gamma 1 SH2 domain were tyrosine phosphorylated strongly after UV irradiation. A similar dose response was observed for the inhibition by herbimycin A of UV-induced calcium signals and UV-induced tyrosine phosphorylation of PLC gamma 1 and associated proteins. We propose that in contrast to CD3/Ti stimulation, UV aberrantly triggers lymphocyte signal transduction pathways by a mechanism that bypasses normal receptor control.

Radiobiological features of fresh leukemic bone marrow progenitor cells in acute lymphoblastic leukemia.

The radiobiological features of leukemic progenitor cells (LPC) freshly obtained from 14 T-lineage acute lymphoblastic leukemia (ALL) and 11 B-lineage ALL patients were evaluated using LPC colony assays. No significant radiobiological differences were observed between T-lineage versus B-lineage ALL LPC. Notably, the D0 values displayed a significant interpatient variation in both groups, indicating a pronounced heterogeneity in the radiation sensitivity of LPC. LPC from some patients were very radioresistant, and in additional experiments using cryopreserved bone marrow blasts, up to 32% of LPC could survive 1600 cGy delivered at 100 cGy/min. In six of 11 T-lineage ALL cases and five of ten B-lineage ALL cases, a distinct initial shoulder was present on the single dose radiation survival curves, providing circumstantial evidence that LPC are able to repair sublethal radiation damage. A greater proportion of LPC survived 400 cGy when the dose was delivered in two fractions instead of a single dose, providing direct evidence that LPC in ALL possess a substantial capacity to repair sublethal radiation damage. The interpatient differences in Dq and recovery factor values indicated a marked heterogeneity in the ability of LPC to repair sublethal radiation damage. Analysis of the dose rate effects on the radiation survival of LPC in four ALL cases suggested that the radiation sensitivity of LPC is dose rate dependent. Normal bone marrow progenitor cells (colony-forming unit, granulocyte-macrophage, and burst-forming unit, erythroid) were more radiosensitive and unable to repair sublethal radiation damage. To our knowledge, this report represents (a) the first detailed comparative analysis of the radiobiological features of freshly obtained LPC in T-lineage and B-lineage ALL patients, and (b) the first elucidation of radiobiological differences between leukemic ALL versus normal bone marrow progenitor cells.

Increased efficiency in selective elimination of leukemia cells by a combination of a stable derivative of cyclophosphamide and a human B-cell-specific immunotoxin containing pokeweed antiviral protein.

Leukemia cells were mixed with normal human bone marrow cells to simulate bone marrow from leukemia patients; the mixture was then treated with a combination of stabilized derivative of cyclophosphamide [Mafosfamide (ASTA Z 7557)] and pokeweed antiviral protein-containing immunotoxin. The ability of this protocol for selective elimination of B-ALL cells was evaluated by clonogenic assay. The monoclonal antibody (B43) portion of the immunotoxin was directed against human B-cells and was linked to pokeweed antiviral protein by a disulfide bond. The combination of ASTA Z 7557 and immunotoxin was superior to either ASTA Z 7557 or the immunotoxin alone and produced nearly 7 logs of elimination of leukemia cells from the cell mixtures. About 5 logs of contaminating tumor cells were eliminated from a 200-fold excess of normal marrow under conditions where fewer than 50% of pluripotent stem cells were lost. Moreover, this manipulation did not inhibit subsequent production of pluripotent stem cells in long-term bone marrow cultures, indicating that the more primitive progenitors were not harmed.

Role of tyrosine phosphorylation in radiation-induced activation of c-jun protooncogene in human lymphohematopoietic precursor cells.

We examined the effects of ionizing radiation on c-jun expression in human lymphohematopoietic precursors. Radiation exposure increased the level of c-jun transcripts in a dose- and time-dependent manner, providing direct evidence that ionizing radiation can activate c-jun protooncogene in human lymphohematopoietic precursors. Notable gamma-rays failed to induce c-jun expression in cells pretreated with herbimycin, and the use of cycloheximide did not overcome the inhibitory effects of herbimycin. The lack of c-jun signal in herbimycin-treated cells was not due to nonspecific damage to the distal protein kinase C signaling pathway. Thus, protein tyrosine kinase activation precedes and perhaps mandates radiation-induced activation of c-jun protooncogene expression in human lymphohematopoietic precursors.

Combined immunochemotherapy of human solid tumors in nude mice.

In vivo immunochemotherapy of human solid tumors was studied in a nude mouse model. Large tumors (3 to 6 cm3) were induced by s.c. injection of the acute lymphoblastic leukemia T-cell line CEM. Transient tumor inhibition could be achieved by intratumoral injection of an intact-ricin immunotoxin that specifically recognizes a determinant CD5 (T,p67) expressed on the cell surface. Injection of the in vitro active cyclophosphamide congener mafosfamid had little effect on the progression of tumor growth. A combination regimen of immunotoxin and mafosfamid induced the most dramatic antitumor effect; a 72 to 100% reduction in tumor volume was observed within 3 to 4 days posttreatment. However, tumors relapsed within 5 to 13 days. Persistent, tumor regression was observed only when protocols using successive injections of combined immunotoxin/mafosfamid were used. All seven mice undergoing this treatment had a precipitous decrease in tumor size, and 86% survived greater than 30 days posttreatment. No residual tumor was detectable on Day 30 in five of seven mice. Regression was partly attributed to the selective activity of immunotoxin, since successive injections of an irrelevant control immunotoxin coupled to ricin in combination with mafosfamid did not reduce tumor size. Thus, we have demonstrated that a combination of anticancer chemotherapy and immunotoxin therapy yielded a greater antitumor effect than either therapy alone.

Establishment of a human t(4;11) leukemia in severe combined immunodeficient mice and successful treatment using anti-CD19 (B43)-pokeweed antiviral protein immunotoxin.

Human acute leukemia, with a chromosomal translocation involving chromosomes 4 and 11, t(4;11)(q21;q23), is the most common form of leukemia in infants and responds very poorly to conventional therapy. A human CD19+ mixed-lineage leukemia cell line with a t(4;11)(q21;q23) translocation, RS4;11, disseminated and proliferated in the hematopoietic tissues and other organs of mice with severe combined immunodeficiency in a manner similar to that observed in humans and killed 100% of the animals. The anti-CD19(B43)-pokeweed antiviral protein immunotoxin selectively inhibited clonogenic RS4;11 cells in vitro, markedly reduced the burden of disseminated leukemia of severe combined immunodeficient mice, and, most importantly, resulted in the long-term survival of treated animals. This severe combined immunodeficient mouse model should be useful for the design of more effective treatment strategies for refractory human leukemias.

Radiation damage repair capacity of primary clonogenic blasts in acute lymphoblastic leukemia.

The sublethal radiation damage repair capacity of primary clonogenic blasts [i.e., leukemic progenitor cells (LPC)] from 74 newly diagnosed acute lymphoblastic leukemia (ALL) patients was analyzed using LPC colony assays. We determined the (a) Dq (quasithreshold dose) and n (extrapolation number) values from single dose radiation survival curves constructed according to the single-hit multitarget model of cell survival, (b) beta values and alpha:beta ratios from single dose radiation survival curves constructed according to the linear quadratic model of cell survival, and (c) recovery factor values from survival data in split-dose experiments. Clonogenic blasts from different ALL patients varied substantially in their ability to repair sublethal radiation damage. However, in 11 of 30 (37%) T-lineage ALL cases and 13 of 44 (30%) B-lineage ALL cases, the radiation survival curves of fresh LPC were characterized by a distinct initial shoulder, providing circumstantial evidence that LPC from a significant portion of ALL patients (24 of 74 cases = 32%) are able to repair sublethal radiation damage. Leukemic progenitor cells from 23 of 34 (68%) evaluable cases evaluated had alpha:beta ratios of < 5 Gy, indicating that they may possess a substantial capacity to repair sublethal radiation damage. In order to further elucidate the repair capacity of ALL LPC, we compared the antileukemic efficacy of fractionated irradiation with 2 x 2 Gy to the antileukemic efficacy of single-dose irradiation with 1 x 4 Gy. In 20 of 28 cases (71%), a > or = 20% increase in LPC survival (recovery factor > or = 1.2) was observed when 4 Gy were delivered in two doses instead of a single dose, providing direct evidence that ALL LPC are able to repair sublethal radiation damage. Our results indicate that modifications in total body irradiation regimens, currently administered prior to bone marrow transplantation, may be necessary to diminish the probability of relapse in high-risk ALL.

Development and characterization of three recombinant single chain antibody fragments (scFvs) directed against the CD19 antigen.

Antibodies that recognize antigens restricted to leukemia, lymphoma, and normal hematopoietic cells represent a unique opportunity to develop therapeutics, because they have the potential for relatively selective treatment of these diseases. Antibodies that recognize the CD19 antigen found on normal and malignant B cells, but not on stem cells, have been used to develop immunoconjugates. However, these conjugates are large and might be suboptimal in tumor penetration when compared to molecules using smaller single chain Fv (scFv) antibody fragments. scFv has the advantage of being a molecularly engineered homogeneous molecule. In this report, we demonstrate the cloning, expression, and binding of three anti-CD19 antibodies as scFvs. All three scFvs were successfully cloned and expressed. FVS191, derived from cell line B43, and FVS192, derived from SJ25C1, were properly refolded and bound CD19 antigen in FACS competition assays. These anti-CD19 scFv should be useful in the further development of diagnostic and therapeutic molecules.

In vitro and in vivo cytotoxicity of an anti-osteosarcoma immunotoxin containing pokeweed antiviral protein.

Successful treatment of many patients with osteosarcoma requires more effective chemotherapy. Since new agents are needed, we have developed an immunotoxin using TP-3, an IgG2b mAb which recognizes human and canine osteosarcomas and budding capillaries of tumors. The plant hemitoxin, pokeweed antiviral protein (PAP), was conjugated to TP-3 to produce an immunotoxin highly active against osteosarcoma. After 48 h no viable human OHS osteosarcoma cells were present in cultures containing TP-3-PAP as demonstrated by the absence of [3H]thymidine uptake into DNA. Furthermore, clonogenic assays indicated > 3.9 log kill of OHS at 18 h. The IC50 of TP-3-PAP against OHS was 3.5 +/- 1.0 (SD) x 10(-12) M. TP-3 mAb without PAP had no effect on OHS proliferation; PAP alone had no effect on OHS growth unless concentrations > 1000 pM were used. When TP-3-PAP (1.25 micrograms-10.0 micrograms) was given i.p. q.d. on days 3-5 after tumor inoculation, a dose-dependent reduction of the number of lung metastases was observed (P < 0.001). These results indicate that the TP-3-PAP immunotoxin may be useful in the treatment of osteosarcoma and some soft tissue sarcomas.

Spleen tyrosine kinase (Syk) deficiency in childhood pro-B cell acute lymphoblastic leukemia.

The cytoplasmic spleen tyrosine kinase (SYK) is a key regulator of signal transduction events, apoptosis and orderly cell cycle progression in B-lineage lymphoid cells. Although SYK has not been linked to a human disease, defective expression of the closely related T-cell tyrosine kinase ZAP-70 has been associated with severe combined immunodeficiency. Childhood CD19(+)CD10(-) pro-B cell acute lymphoblastic leukemia (ALL) is thought to originate from B-cell precursors with a maturational arrest at the pro-B cell stage and it is associated with poor prognosis. Since lethally irradiated mice reconstituted with SYK-deficient fetal liver-derived lymphohematopoietic progenitor cells show a block in B-cell ontogeny at the pro-B to pre-B cell transition, we examined the SYK expression profiles of primary leukemic cells from children with pro-B cell ALL. Here we report that leukemic cells from pediatric CD19(+)CD10(-) pro-B cell ALL patients (but not leukemic cells from patients with CD19(+)CD10(+) common pre-pre-B cell ALL) have markedly reduced SYK activity. Sequencing of the reverse transcriptase-polymerase chain reaction (RT-PCR) products of the Syk mRNA in these pro-B leukemia cells revealed profoundly aberrant coding sequences with deletions or insertions. These mRNA species encode abnormal SYK proteins with a missing or truncated catalytic kinase domain. In contrast to pro-B leukemia cells, pre-pre-B leukemia cells from children with CD19(+)CD10(+) common B-lineage ALL and EBV-transformed B-cell lines from healthy volunteers expressed wild-type Syk coding sequences. Examination of the genomic structure of the Syk gene by inter-exonic PCR and genomic cloning demonstrated that the deletions and insertions in the abnormal mRNA species of pro-B leukemia cells are caused by aberrant splicing resulting in either mis-splicing, exon skipping or inclusion of alternative exons, consistent with an abnormal posttranscriptional regulation of alternative splicing of Syk pre-mRNA. Our findings link for the first time specific molecular defects involving the Syk gene to an immunophenotypically distinct category of childhood ALL. To our knowledge, this is the first discovery of a specific tyrosine kinase deficiency in a human hematologic malignancy.

Biased distribution of chromosomal breakpoints involving the MLL gene in infants versus children and adults with t(4;11) ALL.

Derivative chromosomes of 40 patients diagnosed with t(4;11) acute lymphoblastic leukemia (ALL) were analysed on the genomic DNA level. Chromosomal breakpoints were identified in most cases within the known breakpoint cluster regions of the involved MLL and AF4 genes. Due to our current knowledge of the primary DNA sequences of both breakpoint cluster regions, specific features were identified at the chromosomal fusion sites, including deletions, inversions and duplications of parental DNA sequences. After separation of all t(4;11) leukemia patients into two age classes (below and above 1 year of age), the analysis of chromosomal fusion sites revealed significant differences in the distribution of chromosomal breakpoints and led to the definition of two hotspot areas within the MLL breakpoint cluster region. This may point to the possibility of different age-linked mechanisms that were leading to t(4;11) chromosomal translocations.

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.