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As more and more cell and gene therapies are being developed and with the increasing number of regulatory approvals being obtained, there is an emerging and pressing need for industrial translation. Process efficiency, associated cost drivers and regulatory requirements are issues that need to be addressed before industrialisation of cell and gene therapies can be established. Automation has the potential to address these issues and pave the way towards commercialisation and mass production as it has been the case for 'classical' production industries. This review provides an insight into how automation can help address the manufacturing issues arising from the development of large-scale manufacturing processes for modern cell and gene therapy. The existing automated technologies with applicability in cell and gene therapy manufacturing are summarized and evaluated here.

With the availability of chemotherapy agents for first- and second-line treatment of advanced non-small-cell lung cancer (NSCLC), the patient population that requires subsequent chemotherapy is increasing. This retrospective analysis was performed to describe the clinical course after two standard or approved chemotherapy agents in patients with good overall performance status. Data were selected from patients with advanced NSCLC who had received third- or fourth-line chemotherapy after two prior chemotherapy regimens that included platinum and docetaxel given concurrently or sequentially. Prior regiments had failed due to discase progression within 90 days of chemotherapy, or unacceptable toxicity. Examination of over 700 patient records between January 1993 and January 2000 at one US and one European cancer centre revealed 43 patients that fulfilled the inclusion criteria. Response rates decreased with each line of treatment: first line, 20.9%; second line, 16.3%; third line, 2.3%; and fourth line, 0%. The disease control rate (response plus stable disease) also decreased dramatically from first- to fourth-line treatment, although it was higher for second-line treatment (74.4%) than for first-line treatment (62.8%). The median overall survival time from diagnosis was 16.4 months. The median overall survival time from the start of the last treatment (either third or fourth line) was 4 months. Patients with stage III disease at diagnosis had a longer overall survival from diagnosis than patients with stage IV disease (P=0.02). This review highlights the need for novel therapy approaches for patients with recurrent NSCLC who have failed second-line therapy and provides a baseline for the statistical design of such studies.

Background: Hypertrophic cardiomyopathy is a myocardial disorder resulting from inherited sarcomeric dysfunction. We report a mutation in the myosin-binding protein-C (MyBP-C) gene, its clinical consequences in a large family, and myocardial tissue findings that may provide insight into the mechanism of disease., Methods and Results: History and clinical status (examination, ECG, and echocardiography) were assessed in 49 members of a multigeneration family. Linkage analysis implicated the MyBP-C gene on chromosome 11. Myocardial mRNA, genomic MyBP-C DNA, and the myocardial proteins of patients and healthy relatives were analyzed. A single guanine nucleotide insertion in exon 25 of the MyBP-C gene resulted in the loss of 40 bases in abnormally processed mRNA. A 30-kDa truncation at the C-terminus of the protein was predicted, but a polypeptide of the expected size ( approximately 95 kDa) was not detected by immunoblot testing. The disease phenotype in this family was characterized in detail: only 10 of 27 gene carriers fulfilled diagnostic criteria. Five carriers showed borderline hypertrophic cardiomyopathy, and 12 carriers were asymptomatic, with normal ECG and echocardiograms. The age of onset in symptomatic patients was late (29 to 68 years). In 2 patients, outflow obstruction required surgery. Two family members experienced premature sudden cardiac death, but survival at 50 years was 95%., Conclusions: Penetrance of this mutation was incomplete and age-dependent. The large number of asymptomatic carriers and the good prognosis support the interpretation of benign disease.

Purpose: Despite improved event-free survival of older children with acute lymphocytic leukemia (ALL), infants <1 year of age continue to have a very poor prognosis. A new therapy designed specifically for infants with ALL was initiated., Patients and Methods: From 1984 until 1990, 82 eligible infants <1 year of age were entered on a Pediatric Oncology Group (POG) protocol 8493 for infant ALL. Compared to older patients, infants at diagnosis had more overt CNS leukemia (26%), higher initial WBC count (56% >50,000/microl), and a higher likelihood of CD-10 (CALLA) negative lymphoblasts (55%). A translocation involving chromosome 11 at band q23 was detected in 27 of 64 cytogenetically informative cases. Treatment was based upon two institutional pilot studies utilizing chemotherapy doses based upon body weight. Important components included remission induction with cyclophosphamide (Ctx), vincristine (Vcr), cytosine arabinoside (Ara-C), and prednisone (Pred) (COAP); consolidation therapy with teniposide (VM-26) and Ara-C; and continuation therapy with alternating pulses of COAP with VM-26/Ara-C separated by a methotrexate (Mtx) and 6-mercaptopurine (6-MP) backbone plus CNS therapy consisting of standard triple intrathecal therapy (TIT) (Mtx/hydrocortisone/Ara-C), which avoided the use of radiotherapy in this population., Results: Seventy-six infants achieved a complete remission (93%). Fifty patients have relapsed: 35 isolated marrow relapses, five isolated CNS relapses, eight combined marrow and CNS relapses, and two other relapses. Actuarial event-free survival was 28% (SE = 5%) at 4 years. Infants >274 days (9 months) at diagnosis had a better outcome than those <274 days., Conclusions: This study represents a modest outcome improvement in comparison to previous experience with ALL for infants treated on POG trials. More effective therapy is still needed for infants with ALL.

Background: It is unclear how best to treat children with acute lymphoblastic leukemia who are in a second remission after a bone marrow relapse. For those with HLA-identical siblings, the question of whether to perform a bone marrow transplantation or to continue chemotherapy has not been answered., Methods: We compared the results of treatment with marrow transplants from HLA-identical siblings in 376 children, as reported to the International Bone Marrow Transplant Registry, with the results of chemotherapy in 540 children treated by the Pediatric Oncology Group. A preliminary analysis identified variables associated with treatment failure in both groups. We selected cohorts by matching these variables. A possible bias associated with differences in the interval between remission and treatment was controlled for by choosing matched pairs in which the duration of the second remission in the chemotherapy recipient was at least as long as the time between the second remission and transplantation in the transplant recipient. A total of 255 matched pairs were studied., Results: The mean (+/- SE) probability of a relapse at five years was significantly lower among the transplant recipients than among the chemotherapy recipients (45 +/- 4 percent vs. 80 +/- 3 percent, P < 0.001). At five years the probability of leukemia-free survival was higher after transplantation than after chemotherapy (40 +/- 3 percent vs. 17 +/- 3 percent, P < 0.001). The relative benefit of transplantation as compared with chemotherapy was similar in children with prognostic factors indicating a high or low risk of relapse (the duration of the first remission, age, leukocyte count at the time of the diagnosis, and phenotype of the leukemic cells)., Conclusions: For children with acute lymphoblastic leukemia in a second remission, bone marrow transplants from HLA-identical siblings result in fewer relapses and longer leukemia-free survival than does chemotherapy.

Background: Children with chemotherapy refractory T-cell lymphoblastic leukemia/lymphoma were given alpha-interferon (alpha-IFN) to evaluate the efficacy and toxicity of this biologic response modifier., Methods: Twenty children with T-cell acute lymphoblastic leukemia (T-cell ALL) in marrow relapse and one patient with mediastinal recurrence of T-cell non-Hodgkin's lymphoma (T-cell NHL) were enrolled. All patients had failed at least two previous multiagent drug trials. Recombinant alpha-IFN was given at 30 million U/M2/dose intravenously or subcutaneously for 10 doses over 14 days, followed by 3 doses per week until disease progression occurred., Results: One child had a complete response (< 5% blasts) and three patients a partial response (5-25% blasts) in their bone marrow. All patients eventually showed signs of progressive disease. Significant toxicities included cardiac hypofunction in two patients and profound lethargy in two patients., Conclusions: alpha-IFN is tolerated in children with T-cell ALL and T-cell NHL and has activity against chemotherapy resistant disease.

Childhood ALL has provided the model for basic therapeutic principles in the past and now provides the model for late effects studies. Common threads which run throughout the literature in this area of clinical research are the importance of young age with increased vulnerability to long-term treatment induced sequelae and the relatively large contribution of radiation as compared with chemotherapy in the pathogenesis of adverse sequelae. Previous retrospective studies of long-term childhood ALL survivors focused on neuropsychologic changes and anatomic changes in the CNS after cranial irradiation. More recent retrospective studies have made the following new observations: (i) the high frequency of significant short adult stature in those less than 6 years of age at diagnosis who received 24 Gy cranial irradiation; (ii) actuarial risk of 2.5% of developing a second malignancy with approximately one-half of secondary malignancies occurring in the CNS in children 5 years of age or less who received cranial irradiation; (iii) the association of secondary ANLL with epipodophyllo-toxin use, and (iv) delayed cardiac toxicity despite anthracycline dosage reduction. Current therapy regimens, especially in high-risk patients, are both more successful and more intensive than those used in the past. While it will be another decade before many of the long-term sequelae begin to emerge, one can anticipate, based on current experience, some of the problems that will occur.

Purpose: To determine the dose-limiting toxicity and potential efficacy of topotecan in pediatric patients with refractory malignant solid tumors., Patients and Methods: In this phase I clinical trial, 27 patients received topotecan 0.75-1.9 mg/m2 by continuous intravenous infusion daily for 3 days. Fifty-three treatment courses were given to these patients., Results: Myelosuppression was the dose-limiting toxicity at levels of 1.3 to 1.9 mg/m2 for 3 days, requiring significant support with transfused packed RBCs and platelets. Myelosuppression was variable in severity at the 1.0-mg/m2 dosage level; thus, additional patients were treated with this dosage, followed by human recombinant granulocyte-colony stimulating factor (G-CSF). Other toxicities were not significant. One patient with neuroblastoma had a complete response that lasted for 8 months. Stable disease activity was recorded for other patients with neuroblastoma, rhabdomyosarcoma, and islet cell carcinoma. Pharmacokinetic studies showed that topotecan plasma concentrations ranged from 1.6 to 7.5 ng/mL during infusions of 1.0 mg/m2/d, and that there was a biphasic plasma distribution with a mean terminal half-life of 2.9 +2- 1.0 hours., Conclusion: Topotecan is a promising anticancer agent that deserves phase II testing in pediatric solid tumors. We recommend that pediatric phase II topotecan trials use 1.0 mg/m2/d for 3 days as a constant intravenous infusion, followed by G-CSF for 14 days, and that these treatment courses be repeated every 21 days.

We report the magnetic resonance imaging (MRI) and clinico-histologic characterization of dorsally exophytic brain stem gliomas (DEBSGs). Between 1983 and 1991, 12 of 51 patients evaluated for the diagnosis of brain stem glioma were found to have DEBSGs emanating from the pons, pontomedullary junction or medulla. Eleven of the 12 patients had classic juvenile pilocytic astrocytomas. Unlike most other brain stem tumors, these patients were young (median 38 months, range 17-75), had a relatively long duration of symptoms (median 7 months, range 2-24) and displayed signs of increased intracranial pressure with limited cranial nerve paresis, absence of pyramidal tract findings, and near normal brain stem auditory-evoked potentials. MRI characteristically showed sharply demarcated lesions with decreased signal intensity on T1, and increased intensity on T2 sequences. Except for cystic areas, these tumors showed bright, uniform enhancement after gadolinium-DTPA. In all patients, 50-100% of the tumor volume could be resected. Three of 10 patients who received no immediate postoperative treatment eventually demonstrated disease progression, and 2 patients with subtotal resections who were treated with radiation and/or chemotherapy postoperatively remain disease-free for extended periods of time. The only death occurred in the 1 patient treated with chemotherapy who died of secondary leukemia. The overall and progression-free survival of these patients at 2 years is 100 and 67% as compared to 18 and 21%, respectively, for other concomitantly treated nonexophytic brain stem gliomas.2+ the ability to achieve significant degrees of resection.

Purpose: Evaluation of high-dose chemotherapy with autologous bone marrow rescue (ABMR) in pediatric malignant gliomas., Patients and Methods: Newly diagnosed (n = 11) and recurrent (n = 2) malignant glioma patients received high-dose chemotherapy within 4 weeks of surgery; three had near total and 10 had subtotal resection/biopsy. High-dose thiotepa (300 mg/m2) and cyclophosphamide (2 g/m2) daily for 3 days were followed by ABMR; response was evaluated at day 30. At day 60, patients with at least stable disease received hyperfractionated (n = 9) or conventional external-beam radiotherapy (n = 2) preceded by local radioactive iodine 125 implantation (n = 2) or radiosurgery (n = 1)., Results: Grade III and IV toxicities after ABMR consisted of mucositis (n = 12), cardiomyopathy (n = 1), acute abdomen (n = 1), pneumonitis (n = 2), and infection (n = 2). One complete and three partial responses were observed; the objective response rate was 31% (95% confidence interval, 9% to 61%). Seven had stable disease, one had disease progression, and one died of toxicity before response evaluation. The median overall and progression-free survival durations after combined modality therapy were 14 months (range, 4 to 30+) and 9 months (range, 0 to 30+), respectively. One patient remains progression-free at 30+ months. Radionecrosis and white matter changes occurred in three patients: one after hyperfractionated irradiation, and two after 125I implants., Conclusion: For patients with bulky residual disease after surgery, survival with this aggressive chemotherapy and radiation regimen is not better than that reported for conventional treatment regimens.

Background: The authors conducted a Phase I dose escalation trial of ifosfamide given daily for 3 consecutive days to 29 children with malignant solid tumors. Twenty-eight of these children had received prior chemotherapy., Methods: Patients were assigned to dosage cohorts separately on the basis of prior exposure to the platinum alkylating agents cisplatin or carboplatin (n = 20) or the absence of such exposure (n = 9). At least three patients in each category were treated at a starting dosage of 2133 mg/m2/d for 3 days. This dosage represented 80% of the total dose delivered in the prior study of ifosfamide given daily over 5 days with dosage escalation of 20% in subsequent cohorts., Results: Myelosuppression was dose-limiting at the second dosage level (2560 mg/m2/d) for patients previously treated with platinum and at the third dosage level (3072 mg/m2/d) for those not previously treated with platinum. Dose-limiting neurotoxicity was seen at 2560 mg/m2/d for the former group, but was not encountered in the latter group., Conclusions: Delivery of ifosfamide daily for 3 days is feasible and safe at recommended dosages of 2133 mg/m2/d for children with prior exposure to platinum and 3000 mg/m2/d for those without prior exposure.

The e antigen of the hepatitis B virus (HBeAg) was expressed in S. cerevisiae. Yeast-derived HBeAg exhibits high HBe antigenicity while lacking any HBc antigenicity. The production yield of HBeAg expressed in yeast is dependent on the host strains and the nature of the leader sequences used in the plasmid constructions. The recombinant antigen is not secreted into the medium, independent from the leader sequences which are used. A simple extraction procedure was developed, enabling the isolation of HBeAg from the cells without killing them. Recombinant HBeAg derived from yeast can replace plasma-derived antigen in ELISA for determining antibodies to HBeAg.

This study assessed the association of young age at treatment, cranial irradiation, and time since treatment with intellectual deterioration among 49 long-term survivors of childhood leukemia. Ss had been randomized to receive low-dose cranial radiation therapy or high-dose chemotherapy. Longitudinal assessments of intellect were conducted. No significant effects of treatment group or age at treatment were detected. A small but statistically significant decline in mean full-scale IQ was noted over time (M = -3.6). Reanalysis with IQ test version included as a covariate eliminated IQ declines found initially. Results suggest (a) that there has been reduced toxicity of these methods of treatment and (b) that the magnitude and direction of error introduced by changing tests may approximate the magnitude of adverse effects on IQ from treatment.

Long-term follow-up observations are reported on 427 patients who received one of three different intensified therapies in total therapy study X for acute lymphoblastic leukemia (ALL). In the trial for 'standard-risk' ALL, 154 of 309 patients in complete remission were randomized to receive high-dose methotrexate (HDMTX, 1 g/m2) periodically during the first 72 of 120 weeks of standard continuation therapy with 6-mercaptopurine and oral MTX; the remaining 155 patients received 1800 cGy cranial irradiation and intrathecal MTX, followed by 6-mercaptopurine/MTX therapy interrupted from week 36-71 for substitution of two other pairs of drugs. At 9 years of follow-up, significantly higher proportions of patients in the HDMTX group have maintained complete remissions (64 +/- 7%, SE, vs. 52 +/- 6%, p = 0.03), hematologic remissions (73 +/- 6% vs. 62 +/- 6%, p = 0.03), and testicular remissions (94 +/- 5% vs. 80 +/- 8%, p = 0.03); however, the proportion continuing in central nervous system remission has been lower (84 +/- 5% vs 93 +/- 4%, p = 0.02). In the evaluation of teniposide/cytarabine and delayed cranial irradiation for 'high-risk' ALL, 36 +/- 9% of 101 patients are predicted to be event-free survivors at 9 years. Altogether, 217 (51%) of the 427 patients are event-free survivors after at least 7 years of follow-up (median, 9 years); an additional 75 patients are alive and free of leukemia for a median of 6.4 years after successful remission retrieval therapy, boosting the total number of long-term survivors to 292 (68%). These results establish the efficacy of HDMTX for patients with standard-risk ALL and indicate the potential of teniposide/cytarabine for use in multiagent regimens for patients with high-risk disease. The overall survival figure, 68%, affords a benchmark for other studies assessing long-term outcome in ALL.

The therapeutic efficacy and toxicity of alpha-interferon (alpha-IFN) (Roferon, Hoffmann-La Roche, Inc., Nutley, NJ) were determined in 15 children (age range, 6 to 20 years) with Philadelphia chromosome-positive chronic myelocytic leukemia (Ph+ CML). All patients had received cytoreductive therapy with either hydroxyurea (n = 13) or busulfan (n = 1) or both (n = 1) for 6 weeks to 46 months (median, 7 months) before beginning alpha-IFN therapy at a dose of 5 x 10(6) U/m2/d intramuscularly. This dose was escalated to 10 x 10(6) U/m2/d if leukemia was inadequately controlled. Ten children had a hematologic response, with nine showing a reduction in the percentage of Ph+ marrow cells, including four who had no detectable Ph+ cells in marrow samples collected 48 to 204 weeks after the initiation of therapy. Two of 15 patients remain free of Ph+ cells. Therapy was discontinued before week 104 in ten patients because of the following: (1) early hematologic responses without a decrease in Ph+ cells (three patients); (2) early resistant disease (one patient); (3) blast crisis (one patient); (4) progressive disease (two patients); (5) seizure attributed to high-dose alpha-IFN (one patient); or (6) an inadequate trial of alpha-IFN caused by aseptic necrosis or poor compliance (two patients). The most common side effects were mild and have included fever, malaise, headache, myalgias, and pain at the injection site. Adverse events causing interruption of therapy were seizures, aseptic necrosis, and myelofibrosis. alpha-IFN stabilizes the chronic phase of Ph+ CML in some children, is adequately tolerated when administered at a dose of 2.5 to 5 x 10(6) U/m2/d intramuscularly, and results in a significant decrease in the proportion of Ph+ metaphases in some patients. alpha-IFN in combination with an effective cytoreductive agent or agents appears worthy of further clinical testing in this disease.

We studied the frequency, causes, and predictors of adverse events in 624 patients who had completed treatment for acute lymphoblastic leukemia (ALL) in three consecutive total therapy studies (VII, IX, and X, 1972 to 1983). Event-free survival in study X was significantly better overall than that in studies VIII and IX (P less than .0001 by the log-rank test). In study X, 75% of the patients were electively taken off therapy, compared with 54% in studies VIII and IX. However, the risks of having an adverse event during the first 5 years after completion of therapy were remarkably similar: 22% (95% confidence interval, 17% to 29%) in study X versus 24% (20% to 29%) in studies VIII and IX. Bone marrow, testicular, and CNS relapses accounted for the majority of failures in both groups (85% in study X and 92% in studies VIII and IX). Late adverse events consisted largely of hematologic relapses and the development of solid tumors. Black race (P = .001) and leukemia without an anterior mediastinal mass (P = .05) were associated with an increased risk of failure after completion of treatment in the two earlier clinical trials, whereas a lower leukemic cell DNA content (DNA index less than 1.16) was the only predictor of late treatment failure in the more recent trial (P = .019). None of the other presenting features that were examined (eg, age, leukocyte count, and sex) had value as predictors of late failure. Thus, improved treatment altered the impact of specific prognostic factors and the distribution of sites of relapse, but it did not significantly affect the risk of delayed failure.

To compare the late neuropsychologic toxicities of CNS prophylaxis for childhood acute lymphoblastic leukemia (ALL), longitudinal assessments were performed on three groups of patients: those who received repeated courses of moderate-dose (1 g/m2) intravenous (IV) and intrathecal methotrexate (IT MTX) without cranial irradiation (MTX group, n = 26), those who received IT MTX and 18 Gy cranial irradiation (18-Gy group, n = 23), and those who received IT MTX and 24 Gy cranial irradiation (24-Gy group, n = 28). All patients were free of CNS leukemia at diagnosis and had remained in continuous, complete remission 5 to 11 years (median, 7.4 years) following CNS prophylaxis. An analysis of serial intelligence quotient (IQ), achievement, and neuropsychologic studies revealed no significant influence of either age at CNS prophylaxis or CNS prophylaxis group on any neuropsychologic outcome measure. After adjusting for changes in IQ test versions that were necessitated by advancing patient age, no statistically significant declines in Verbal, Performance, or Full Scale IQs were noted for the three CNS treatment groups. However, comparisons of group means masked declines in individual children; 22% to 30% of children exhibited a clinically significant deterioration (greater than or equal to 15 points) in uncorrected IQ values over the study period. Female sex was associated with an increased risk of deterioration in Verbal IQ, but we were unable to identify risk factors associated with other declines in intellect and achievement. The inability to reliably predict individual patients at risk for clinically significant neuropsychologic toxicities on the basis of age at diagnosis or specific method of CNS prophylaxis suggests that other etiologic factors must be explored as the basis for these changes, such as ecologic factors and chemotherapy during the continuation phase of treatment.

Recombinant interferon alfa (rIFN-alpha) was given to 31 children with acute lymphoblastic leukemia (ALL) in first on-therapy marrow relapse as the sole treatment (30 megaunits/m2/d intravenously x 10 days) before standard four-drug reinduction and during multiagent continuation therapy (30 megaunits/m2 subcutaneously x 3 consecutive days every 3 weeks). After 10 days of rIFN-alpha, there were two partial remissions (PRs); seven additional patients had either greater than or equal to 25% reduction in the percentage of marrow blast cells or hypoplastic marrow. Two patients had progressive disease with an increase in leukocyte counts. All patients experienced influenza-like symptoms, and there were isolated instances of severe abdominal pain and personality change. Dose-limiting toxicity comprised grade III/IV transaminase elevation (two patients) and syncope with personality change (one patient). Twenty-three of 31 children (74%) subsequently achieved marrow remission using standard agents. One patient was taken off study during teniposide (VM-26) and cytarabine (ara-C) consolidation due to toxicity. Continuation therapy including rIFN-alpha pulse was well tolerated in the remaining children; only one patient required rIFN-alpha dosage reduction (for CNS toxicity). rIFN-alpha toxicity did not necessitate reductions in doses of standard chemotherapy agents or significant delays in therapy. Five patients remain in remission at 26+ to 36+ months; 13 patients relapsed in marrow, one in the meninges (7 months), and one in meninges, mediastinum, and lymph nodes (2 months). Two children were removed from study for marrow transplant. In summary, high-dose rIFN-alpha alone had a modest antileukemic effect. In contrast to the clinical experience with combined rIFN-alpha and chemotherapy in adults, rIFN-alpha given in a pulse-like manner throughout continuation therapy did not compromise the intensity of the standard chemotherapy regimen.

The human blood coagulation protein Factor XIIIa (FXIIIa) was expressed in Saccharomyces cerevisiae employing Escherichia coli-yeast shuttle vectors based on a 2-mu plasmid. Several factors affecting high production yield of recombinant FXIIIa were analysed. The use of the regulatable GAL-CYC1 hybrid promoter resulted in higher FXIIIa expression when compared with the constitutive ADCI promoter. Screening for suitable yeast strains for expression of FXIIIa under the transcriptional control of the GAL-CYC1 hybrid promoter revealed a broad spectrum of productivity. No obvious correlation between the expression rate and the genetic markers of the strains could be identified. The medium composition markedly influenced the FXIIIa expression rates. The expression of FXIIIa was strictly regulated by the carbon source. Glucose as the only sugar and energy source repressed the synthesis of FXIIIa, whereas addition of galactose induced FXIIIa expression. Special feeding schemes resulted in a productivity of up to 100 mg FXIIIa/l in shake flasks.

Compared ratings of everyday cognitive functioning made by parents of leukemic children to ratings made by parents of normal control and learning disabled (LD) children. The leukemic children had been randomly assigned to one of two CNS prophylaxis treatments, one including cranial irradiation and intrathecal methotrexate and another including only intrathecal methotrexate and intermediate dose infusions of methotrexate. Leukemic children were rated significantly worse than controls in areas related to schooling and academic skills. The type of CNS prophylaxis was not discriminated by parent ratings. LD children were rated as significantly worse than either of the two groups in all areas of cognitive functioning. Leukemic and LD children were both rated as having poor academic skills. Leukemic children missed significantly more school than control and LD children, and their poor ratings on academic skills were partially attributed to academic deprivation. These results suggest that studies should control for academic deprivation when evaluating the neuropsychological outcome of CNS prophylactic treatment and that reintegration and normalization programs should be designed to address the intellectual problems resulting from missed academic experiences.

We prospectively compared neuropsychologic functioning and clinical indicators of neurotoxicity in 49 consecutive childhood leukemia patients in long-term continuous complete remission (CR) who had received two different regimens of CNS prophylaxis by random assignment. Twenty-three patients were treated with 1,800 cGy cranial radiation and intrathecal methotrexate (RT group) and 26 with parenteral methotrexate only (MTX group). Over half of the RT group had somnolence syndrome, and four developed cerebral calcifications late in their clinical course. Abnormal electroencephalograms (EEGs) were seen in 15 patients in the MTX group, and six had early, transient white-matter hypodensities apparent on computed tomographic (CT) scans. Mean scores on standard tests of intelligence and academic achievement, administered after remission induction and again at a median of 6 years after treatment cessation, did not differ significantly between the two groups. However, statistically significant decreases in overall and verbal intelligence quotients (IQs) and in arithmetic achievement were found within both treatment groups. Sixteen of 26 in the MTX group and 14 of the 23 in the RT group had clinically important decreases (greater than or equal to 15 points) on one or more neuropsychologic measures. These changes did not correlate with findings on CT scans, EEGs, or other clinical signs of neurotoxicity. We conclude that 1,800 cGy cranial radiation and parenteral methotrexate, as used in this study, are associated with comparable decreases in neuropsychologic function.

Teniposide (VM-26) can increase intracellular methotrexate (MTX) and its polyglutamate derivatives in vitro and thus has the potential to improve the therapeutic index of regimens containing MTX. In this phase II study, children and adolescents with acute lymphoblastic leukemia (ALL) in first or second marrow relapse were randomly assigned to receive either simultaneous (n = 11) or sequential (n = 12) continuous infusions of MTX and VM-26 prior to reinduction. Infusions of VM-26 were begun 12 hours after completion of MTX infusion in the sequential group. Dosages were individually adjusted to maintain plasma concentration levels of 10 microns for MTX and 15 microns for VM-26; total infusion times were 24 and 72 hours, respectively. Significant toxicity in the first six patients who received the scheduled 72-hour VM-26 infusion (including one drug-related death) prompted a 50% reduction in infusion duration. The reduced dose was associated with similar but more manageable toxicity. Examination of bone marrow aspirates 10 days after therapy was begun showed one complete and two partial marrow remissions; a fourth patient who had an aplastic marrow on day 10 received no further chemotherapy and had a complete remission (CR) documented on day 31. There was no obvious clinical advantage associated with either infusion schedule, although small sample sizes preclude definitive conclusions. The 17% response rate to the MTX/VM-26 therapeutic window in patients with refractory disease suggests the need for further investigation to evaluate alternative schedules and concomitant therapy for this drug combination.

Twenty-six evaluable children with newly diagnosed acute lymphoblastic leukemia (ALL) who failed to achieve initial remission after receiving two to seven drugs for at least a 4-week period were given teniposide (VM-26) and continuous infusion cytosine arabinoside (Ara-C). Twenty-two received 150 mg/m2 of VM-26 on days 1 and 2 with 100 mg/2 of Ara-C as a continuous infusion on days 1 through 5; a second shortened course was given on day 14 to eight patients who had evidence of some antileukemic effect or were clinically judged able to tolerate a second course. The last four patients received three daily doses of VM-26 and a 7-day infusion of Ara-C at the same daily dosages. Twelve (48%) achieved complete remission (CR) of ALL. There was a trend toward decreasing response rates with an increasing number of drugs used in the initial induction regimen, i.e., five CR among seven patients with a prior two-drug induction attempt, six CR among 14 patients with a prior three- to four-drug induction attempt, and one CR among four patients with a prior five- to seven-drug induction attempt (P = 0.14). Ten of 17 non-T-cell patients and two of nine T-cell patients achieved remission (P = 0.10). The median time required to achieve a complete remission from the initiation of treatment was 26 days (range, 14-72 days). This period was shorter in those who required one course compared with those who required two induction courses, i.e., 25 days median vs. 44 days median. Toxicity was significant and due mainly to marrow aplasia and infection; one patient had severe prolonged VM-26-induced hypotension. Of the 12 patients entering remission, two were removed for marrow transplant and one was removed due to parental request. In the remaining nine patients, median remission duration was only 2 months (range, 1-18 months). All nine patients relapsed in the marrow. Among the entire group of 26 patients, only one patient is alive and a long-term event-free survivor (after allogeneic marrow transplant). Due to the current use of more aggressive initial induction regimens and the extremely poor prognosis in children who fail to achieve initial remission, more intensive regimens of continuation therapy or alternative therapies, such as bone marrow transplant, should be considered.

Laboratory studies have demonstrated the ability of teniposide to markedly enhance the intracellular accumulation of methotrexate suggesting that combination therapy with these agents may produce clinical benefit. Studies of methotrexate and teniposide were conducted in 19 children with relapsed acute lymphocytic leukemia to evaluate the pharmacokinetics of this previously untested combination of agents given alone or in combination and to demonstrate the feasibility of a Bayesian dose optimization strategy. Patients were randomly assigned to receive intermediate dose methotrexate as a 24-h continuous infusion, administered either simultaneously with continuous infusion teniposide or sequentially with the teniposide infusion beginning 12 h after the end of the methotrexate infusion. Plasma samples were obtained during and after infusions at appropriate times for a comprehensive pharmacokinetic study of each drug. Two measured drug concentrations obtained during the infusion were used to adjust each patient's dose rate to achieve target values of 10 microM for methotrexate and 15 microM for teniposide. Pharmacokinetic parameters for teniposide were not different for patients given simultaneous methotrexate from parameters estimated for patients receiving teniposide 12 h after the end of the methotrexate infusion. Despite similar end of infusion methotrexate concentrations, 24-h postinfusion methotrexate concentrations were lower (0.137 versus 0.235 microM; P less than 0.05) in the patients receiving simultaneous infusions. The patient specific dose regimens yielded acceptably precise, minimally biased steady state drug concentrations. These pharmacokinetic results provide the basis for further clinical studies with this combination of antileukemic agents.

Four patients with acute nonlymphocytic leukemia who were heterozygous for the X-chromosome-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) were studied to determine the numbers and types of progenitor cells in which the disease arose. Both forms of enzyme were found in normal tissues, but the malignant blast cells showed only one G6PD, indicating that the disease was clonal at the time of testing. The observations that normal erythroid cells were present in two young patients at diagnosis and relapse indicate that the clone suppressed expression of normal granulopoiesis but did not prevent normal erythroid differentiation. In contrast to this situation, in two elderly patients, the disease involved stem cells multipotent for granulocytes, red cells, and platelets. These results indicate that acute nonlymphocytic leukemia is heterogeneous. In some patients, the disease is expressed in cells with differentiation restricted to the granulocyte-monocyte pathway; in others, it involves stem cells capable of differentiating to granulocytes-monocytes, platelets, and erythrocytes. This heterogeneity may reflect differences in causation and could have prognostic and therapeutic importance.

Increasing numbers of childhood ALL survivors have increased the need to assess the physical and psychosocial functioning of this group in a careful manner. This article reviews data on the frequency and types of second malignancies, structural and functional changes in the central nervous system, endocrine effects on growth and reproduction, and psychosocial aspects of development. Most long-term survivors of ALL do not have serious or life-threatening medical problems; however, medical and psychosocial problems may not be insignificant and may require coordinated management over prolonged periods.

High-dose methotrexate (HDMTX) added to a basic regimen of chemotherapy proved superior to cranial irradiation and sequentially administered drug pairs (RTSC) in prolonging complete remissions in children with "standard-risk" acute lymphocytic leukemia. To extend this result to more contemporary risk groups, we reclassified the patients according to methods of the Pediatric Oncology Group (POG), the Childrens Cancer Study Group (CCG), the Rome workshop, and St Jude Total Therapy Study XI. By life table analysis, 70% to 78% of patients with a favorable prognosis would remain in continuous complete remission (CCR) at 4 years if treated with HDMTX. Uniformly lower CCR rates could be expected with RTSC, especially in St Jude better-risk patients. HDMTX also would show greater efficacy than RTSC in the CCG average-risk and POG poor-risk groups, but the results appear inferior to those being achieved with intensified regimens for high-risk leukemia. Although both therapies would provide adequate CNS prophylaxis in favorable-risk groups, RTSC would offer greater protection in patients classified as being in a worse-risk group by St Jude criteria. We conclude that HDMTX-based therapy, as described in this report, would be most effective in patients with a presenting leukocyte count of less than 25 x 10(9)/L, of the white race, aged 2 to 10 years, and having leukemic cell hyperdiploidy without translocations.

Using flow cytometric techniques, we determined the pretreatment distribution of DNA content in propidium iodide-stained leukemic blasts from 205 children with "standard-risk" acute lymphoblastic leukemia (ALL). Risk assignment was based on an initial WBC count less than 100 X 10(9)/L, no thymic mass, no meningeal leukemia, and lymphoblasts lacking sheep erythrocyte receptors or surface immunoglobulin. A single aneuploid leukemic line was detected in 74 cases (36.1%): 70 hyperdiploid and four hypodiploid. For hyperdiploid cases, the DNA index (DI, or ratio of the DNA content of leukemic v normal G0/G1 cells) ranged from 1.06 to 2.0 (median, 1.20). A secondary leukemic line with hyperdiploid cellular DNA content was identified in 21 cases with diploid primary lines. Children whose primary leukemic line showed a DI greater than or equal to 1.16 (n = 57) had significantly better responses to treatment than did those with either a diploid DI (n = 130; P = .002) or values in the range of 1.01 to 1.15 (n = 14; P = .001). The relative risk of failure for hyperdiploid cases with DI greater than or equal to 1.16, corresponding to greater than or equal to 53 chromosomes, was one-third that of the other two groups. Treatment responses of patients with both diploid and hyperdiploid lines were identical to those associated with single diploid lines, but significantly worse than those associated with single hyperdiploid lines with DI greater than or equal to 1.16 (P = .016). The most favorable prognostic variables selected by a Cox proportional hazards model were: DI greater than or equal to 1.16 (P = .001), white race (P = .022), WBC less than or equal to 25 X 10(9)/L (P = .032), age between 2 and 9 years (P = .075), and hemoglobin less than 7.0 g/dL (P = .094). DNA index greater than or equal to 1.16 retained its significant prognostic impact even after adjustment for other variables (P = .001). With the combination of DI greater than or equal to 1.16 and WBC less than or equal to 25 X 10(9)/L, one can identify a group of children with ALL who have a low probability of relapse when treated with current therapy. If they remain disease-free after longer follow-up, it may be advisable to treat them with less intensive, hence less toxic, chemotherapy than patients with higher WBC counts or lower DI values.

Analyses of bone marrow blast cells collected at diagnosis and relapse from 68 children with acute lymphoblastic leukemia (ALL) demonstrated changes in the expression of cell markers in one-fourth of the patients. Loss of the common ALL antigen (CALLA) was a frequent change, occurring in 8 of the 51 cases initially classified as common or pre-B ALL. The HLA-DR antigen was either acquired or lost in 5 of the 68 cases, terminal deoxynucleotidyl transferase was lost in 6 of 25 cases, and reactivity of the T10 antigen with monoclonal antibodies was increased in 6 of 17 cases of non-T cell ALL. Conversion to acute nonlymphoblastic leukemia, so-called lineage switch, was noted in two cases of common ALL and one of pre-B ALL, coinciding with the loss of CALLA. Results of chromosomal analyses in cases with a loss of CALLA implicated several mechanisms in the observed phenotypic changes. In six cases, including each instance of lineage switch, the original karyotype had been replaced by an entirely different abnormal karyotype, suggesting clonal selection or induction of a second malignancy. In another case, the evidence suggested clonal evolution. Our findings demonstrate that sequential phenotypic and cytogenetic studies may yield valuable insights into the mechanisms of leukemic recurrence and may have implications for treatment selection.

Samples of leukemic cell DNA from 14 children with acute nonlymphocytic leukemia (ANLL) and 4 human myeloid leukemia cell lines were analyzed for rearrangement in the heavy chain region of the immunoglobulin gene. The diagnosis of ANLL was confirmed in all patients by morphological, cytochemical, and immunologic studies. By restriction endonuclease digestion and hybridization with cloned heavy chain immunoglobulin gene probes for the constant (Cmu) and joining (JH) regions, the DNA of 2 patients and 1 cell line (ML-1) was found to contain rearrangements. The DNA from the remaining 12 patients and 3 cell lines was not rearranged (germline configuration). Both patients with apparent immunoglobulin gene rearrangement achieved complete remission on therapy for ANLL. Immunoglobulin gene rearrangement in phenotypically defined ANLL suggests (1) that such changes may not be limited to lymphoid leukemia of B cell lineage, or (2) that, in some patients, the leukemic transforming event may involve stem cells capable of both B cell and myeloid differentiation.