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PURPOSE: A retrospective analysis of the treatment of childhood acute lymphoblastic leukemia (ALL) in second remission (CR2) was undertaken at our institution to compare the outcome and prognostic factors of patients treated with chemotherapy or allogeneic bone marrow transplantation (BMT). PATIENTS AND METHODS: Seventy-five children who suffered a medullary relapse and achieved a second remission were treated with either an unmodified allogeneic HLA-matched sibling BMT after hyperfractionated total body irradiation (TBI) and cyclophosphamide (n = 38) or chemotherapy according to institutional chemotherapy protocols (n = 37). To avoid the bias of survival from the attainment of second remission in favor of BMT, the final comparative statistical analysis used the landmark approach and comprised 37 and 29 patients from the BMT and chemotherapy groups, respectively RESULTS: The disease-free survival (DFS) rate was 62% and 26% at 5 years, respectively, for the BMT and the chemotherapy groups (P = .03), with relapse rates of 19% and 67%, respectively, for these two groups (P = .01). There was an overall advantage for the BMT therapeutic approach, as compared with chemotherapy, for patients with ALL in CR2 (1) for patients with a WBC count (at diagnosis) of 20 × 109/L or higher (DFS, 40% v 0%) and those with a WBC count of less than 20 × 109/L (DFS, 73% v 35%), (2) for patients whose duration of CR1 was less than 24 months (DFS 48% v 9%) and for patients whose duration of CR1 was 24 months or longer (DFS, 81% v 37%) and (3) for patients who were initially treated with intensive regimens incorporating more than five chemotherapy agents (DFS, 57% v 20%) and for patients treated with five agents or fewer (DFS, 72% v 32%). CONCLUSION: In our single-institution series, unmodified HLA-matched allogeneic sibling transplants using hyperfractionated TBI and cyclophosphamide for patients with ALL in CR2 have resulted in superior outcome with a significantly improved probability of DFS and a lower relapse rate, as compared with those for patients treated with chemotherapy, regardless of the duration of first remission, the disease characteristics at diagnosis, or the intensity of prior treatment during first remission.

Children with Philadelphia (Ph) chromosome positive (+) acute lymphoblastic leukemia (ALL) represent a subgroup at very high risk for treatment failure. This study included 1322 children enrolled between 1988 and 1994 on CCG risk-adjusted studies for ALL who had centrally reviewed cytogenetic data. Thirty patients had a t(9;22) and are referred to as Ph+; 1292 were Ph-; 23 of these 30 patients were treated on the CCG-1882 high risk ALL protocol. The event-free survival (EFS) outcome in CCG-1882 was significantly worse for Ph+ compared with Ph- patients, with 4-year estimates of 11.3% (SD = 9.8%) and 73.4% (SD = 2.3%), respectively (p < 0.0001).

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/μL, 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.

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 (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.

We used reverse transcriptase polymerase chain reaction (RT-PCR) assays to examine primary leukemic cells in on-study diagnostic bone marrow specimens from 642 children with newly diagnosed acute lymphoblastic leukemia (ALL) for the expression of MLL-AF4, E2A-PBX1, and BCR-ABL fusion transcripts. All PCR assays were performed centrally in the Children's Cancer Group ALL Biology Reference Laboratory. MLL-AF4 transcript was found in only 0.7% of the study population which excluded infants. E2A-PBX1 transcript was found in 2.5% of the study population and 3.3% of B-precursor cases. Expression was associated with massive hepatomegaly. BCR-ABL transcript was found in 2.3% of cases and correlated with older age, induction failure, and inferior event-free survival (EFS). RT-PCR assays allow rapid identification of patients with MLL-AF4 and BCR-ABL positive ALL. These patients have a poor outcome with contemporary therapy and rapid identification facilitates timely allocation to innovative treatment programs.

Purpose : This study attempted to determine if central nervous system (CNS) disease at diagnosis is a poor prognostic factor in children with acute lymphoblastic leukemia (ALL) and whether 6 Gy of spinal irradiation is an adequate dose for these patients. Methods and Materials : Previously the Children's Cancer Group (CCG) treated patients with ALL and CNS disease at diagnosis with cranio (24 Gy)-spinal (12 Gy) irradiation, as well as systemic and intrathecal chemotherapy. In a series of CCG trials completed in 1989 the spinal dose was empirically reduced to 6 Gy for patients receiving systemic chemotherapy with an intensive consolidation phase to limit hematopoietic toxicity. The spinal dose was left at 12 Gy for patients treated with a less intensive consolidation phase. Results : With a median follow-up for surviving patients of 74 months, the 5-year event-free survival for 53 patients with CNS disease at diagnosis was 69 ± 13% (±2 standard deviations), similar to the value obtained for 3364 patients without CNS disease, 67 ± 2%. Corresponding values for 5-year survival were 77 ± 12% and 80 ± 1%, and for freedom from isolated first CNS relapse, 90 ± 9% and 94 ± 1%. Event-free survival, survival, and freedom from isolated first CNS relapse in the 6-Gy group were as good as in the 12-Gy group. Conclusion : CNS disease at diagnosis is not a poor prognostic factor for children with ALL who are treated with intensive systemic chemotherapy, craniospinal irradiation, and intrathecal chemotherapy. Six Gy is an adequate dose of spinal irradiation for these patients.

PURPOSE To test intensive alkylator-based therapy in desmoplastic small round-cell tumor (DSRCT). PATIENTS AND METHODS Patients received the P6 protocol, which has seven courses of chemotherapy. Courses 1, 2, 3, and 6 included cyclophosphamide 4,200 mg/m2, doxorubicin 75 mg/m2, and vincristine (HD-CAV). Courses 4, 5, and 7 consisted of ifosfamide 9 g/m2 and etoposide 500 mg/m2 for previously untreated patients, or ifosfamide 12 g/m2 and etoposide 1,000 mg/m2 for previously treated patients. Courses started after neutrophil counts reached 500/microL and platelet counts reached 100,000/microL. Tumor resection was attempted. Post-P6 treatment options included radiotherapy and a myeloablative regimen of thiotepa (900 mg/m2) plus carboplatin (1,500 mg/m2), with stem-cell rescue. RESULTS Ten previously untreated and two previously treated patients have completed therapy. The male-to-female ratio was 11:1. Ages were 7 to 22 years (median, 14). The largest masses were infradiaphragmatic (n = 11) or intrathoracic (n = 1). Other findings included serosal implants (n = 11), regional lymph node invasion (n = 8), ascites or pleural effusion (n = 7), and metastases to liver (n = 5), lungs (n = 4), distant lymph nodes (n = 3), spleen (n = 2), and skeleton (n = 2). Tumors uniformly responded to HD-CAV, but there were no complete pathologic responses. One patient died at 1 month from tumor-related Budd-Chiari syndrome. Of seven patients who achieved a complete remission (CR), five remain in CR 9, 12, 13, 33, and 38 months from the start of P6, one patient died of infection at 12 months (autopsy-confirmed CR), and one patient relapsed 4 months off therapy. Of four patients who achieved a partial remission (PR), one remains progression-free at 34 months and three developed progressive disease. Five patients received local radiotherapy: three were not assessable for response, but in two patients, antitumor effect was evident. Four patients received thiotepa/carboplatin: two were in CR and remain so, and two patients had measurable disease that did not respond. CONCLUSION For control of DSRCT, our experience supports intensive use of HD-CAV, aggressive surgery to resect visible disease, radiotherapy to high-risk sites, and myeloablative chemotherapy with stem-cell rescue in selected cases.

PURPOSE To quantify the residual marrow lymphoblast fraction that best defines patients at high risk for relapse, and the optimal time for assessment during remission induction. PATIENTS AND METHODS The residual lymphoblast percentage was evaluated on day 7 (n = 220) and day 14 (n = 205) during a four- or five-drug induction in patients with poor prognostic factors. The rate of cytoreduction was related to event-free survival (EFS) and other factors. RESULTS On the New York (NY) regimen, 68%, 14%, and 18%, and on the Berlin-Frankfurt-Munster (BFM) regimen, 56%, 15%, and 29% of patients had M1 (< 5% blasts), M2 (5-25%), or M3 (> 25%) responses on day 7 (P = .075). On day 14, the corresponding values were 87%, 6%, 7% on NY and 84%, 8%, 8% on BFM. For patients who achieved remission by day 28 and a day-7 marrow rating of M1, M2, or M3, the 6-year EFS rate was 78%, 61%, and 49% (P < .001). The day-14 ratings predicted for a 72%, 32%, or 40% EFS (P < .001). Patients with 5% to 10% blasts day 7 had three times as many events as those with less than 5% and had no better EFS than those with 11% to 25% blasts. Patients with a WBC count more than 200,000/microL at diagnosis and an M1 day 7 marrow had an EFS rate of 69%, while for those with M2 or M3, the EFS rate was 41%. Day-7 marrow had greater prognostic significance than the day-14 evaluation. For slow responders on day 7, the day-14 marrow provided additional information. EFS for patients who achieved M1 by day 14 was 65%. EFS decreased to 20% for those still M2 or M3 on day 14. Day-7 and -14 evaluations had significance for patients of all ages and WBC levels. CONCLUSION Marrow aspiration on day 7 of therapy provided more useful information than that on day 14. However, day-14 marrow provided additional information for patients with a poor day-7 response. The rate of cytoreduction is a powerful, independent prognostic factor that can identify patients with a slow early response who are at risk for a short remission duration.

Examined predictors of depressive symptoms among 59 parents providing primary care to children newly diagnosed with cancer. Parents were studied for a 3-month period. The parent providing primary care to the child during medical treatment completed measures of depressive symptoms, endorsement of family routines, family functioning, amount of assistance from the spouse in providing care to the child, child behavior problems, as well as measures of the severity of the child's treatment regimen. A strong relationship was found between child behavior problems and parent depressive symptomatology. Although disease-related factors such as the child's functional impairment played a role in the parent's depressive symptoms, results revealed that the child's behavior problems were most strongly associated with parent depressive symptoms and that family cohesiveness also had a contributory role in the maintenance of parent depressive symptoms.

Although dihydrofolate reductase (DHFR) gene amplification is a common mechanism of resistance to methotrexate (MTX) in tumor cell lines, with the exception of a few case reports, the incidence of this phenomenon as a mechanism of MTX resistance in the clinic has not been reported. We studied 38 untreated patients and 29 patients in relapse with acute lymphoblastic leukemia (ALL) for gene amplification and p53 gene mutations. Three patients were studied both at diagnosis and at each of two relapses after treatment with MTX. Nine of 29 relapsed patients (31%) had low-level DHFR gene amplification (two to four gene copies) associated with increased levels of DHFR mRNA and enzyme activity. Of significance was a correlation of gene amplification with p53 mutations in seven of nine relapsed patients (P < .001). Low-level DHFR gene amplification may be an important cause of MTX resistance in ALL and strengthens the concept that mutations in the p53 gene may lead to gene amplification as a consequence of defective cell cycle control.

The clinical course of late symptomatic anthracycline cardiomyopathy, and resultant changes of cardiac function, were described in 15 patients. They represented a subset of 300 patients who had cardiac evaluations to identify the prevalence of late cardiotoxicity more than 4 years after anthracycline therapy in these patients. The clinical course and all available cardiac evaluations including electrocardiography, continuous taped electrocardiography, echocardiography, radionuclide cardiac angiography, cardiac catheterization, and endomyocardial biopsy, of the 15 patients were reviewed. The patients had received 285-870 (median 540) mg/M2 of daunorubicin and/or doxorubicin 6-19 (median 12) years prior to the onset of late symptoms. Seven patients also had 2,100-4,000 cGy mediastinal radiotherapy. Five patients had required treatment for cardiac symptoms at the end of chemotherapy but 10 patients had no cardiac problems anteceding their late decompensation. Fractional shortening on echocardiogram at late decompensation was 8-20% (median 17%) and radionuclide left ventricular ejection fraction was 8-59% (median 38%). All were treated with digitalis and diuretics and 13/15 with afterload reduction, with at least transient improvement of symptoms. They were followed for 1-9 (median 3) years after late decompensation. One died of uncontrollable cardiac failure. Another underwent successful cardiac transplantation. Conduction abnormalities and dysrhythmias were present in 14/15 patients and 3 died suddenly. Two more had syncope, one requiring an automatic cardiac defibrillator. Endomyocardial biopsy or autopsy revealed hypertrophy and fibrosis in 10/10 patients. Our patients with early cardiac symptoms improved transiently but decompensated later and patients with no early symptoms developed cardiac symptoms more than 10 years after anthracycline therapy. Therefore, patients who have received anthracyclines should have continued cardiac evaluation.

Abstract 1483 In Vitro culture reveals microenvironment-dependent growth, heterogeneity and hierarchical structure of primary human pediatric pre-B cell acute lymphoblastic leukemia (pre-B ALL) cells. J.-H. Shieh1, P. Steinherz2, J Shieh3 and M. A.S. Moore1. 1Moore Laboratory. Cell Biology Program and 2Leukemia and Lymphoma Studies, Department of Pediatrics, Memorial-Sloan Kettering Cancer Center, New York, NY. 3Department of Biology, Brandeis Univ., Waltham, MA Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children. Although this pediatric pre-B ALLs are treatable, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. To address this issue, we systemically evaluated the effects of various tissue culture parameters to the growth of primary pre-B ALL cells. A serum-free MS-5 cells (a murine bone marrow stromal cell line) co-culture system is capable of expanding the pre-B ALL CD34+CD19+ cells and supporting their differentiation to CD34−CD19+ B cells. This expansion requires a contact between the stromal cells and the pre-B ALL cells, and is inhibited by fetal bovine serum and IL-6 in a dose-dependent manner. c-Kit ligand and Flt3 ligand can reverse the IL-6 inhibition. Expansion of individual CD34+CD19+ cells revealed a hierarchical structure with respect to CD34 antigen expression and an heterogeneity in cell proliferation. When the pre-B ALL cells were sorted into CD34dim and CD34bright populations, the CD34dim cells were capable of a faster proliferation but gradually lost their CD34 antigen. In contrast, the CD34bright cells were more slowly proliferating and retained their CD34 antigen. We transduced the B-ALL cells with a fusion gene expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL). These GFP-Lu-pre-B ALL cells display the similar in vitro characteristics and in vivo xenograftment to NOD/SCID IL2R gamma null (NSG) mice as the non-transduced pre-B ALL cells. One hundred, 103, 104 or 105 GFP-LU-pre-B ALL CD34+ cells were i.v. transplanted to NSG mice. Both 104 and 105 cells resulted in the engraftment of the leukemia cells in limbs and cranium as judged by imaging after 6 weeks, and 103 cells engrafted after 13 weeks. When the 105 cells-transplanted mice were sacrificed after 14 weeks, the harvested peripheral blood, spleen (3–4×108cells/spleen) and bone marrow (5−10×106 cells/femur) displayed 2–3%, 51–55% and 75–81% of human CD34+CD19+ cells, respectively. Human CD34−CD19+ cells were 1–2%, 12–13% and 15–21%, respectively. Therefore, our stromal culture system supports leukemic stem cell/leukemia initiating cell proliferation and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo, and reveals the heterogeneity and hierarchical structure of human pre-B ALL cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides powerful tools to dissect the pathophysiology of human pre-B ALL, and to screen new drugs for pre-B ALL therapy. Disclosures: No relevant conflicts of interest to declare.

6626 Background: AML with t(8;16) represents a rare cytogenetic subtype of adult AML with distinctive morphologic and clinical features. It is associated with very poor prognosis and the molecular pathogenesis of AML is not understood. In several published reports, including the patients reported here, it was initially misdiagnosed and treated as acute promyelocytic leukemia (APL). Furthermore t(8;16) AML is reported to be more frequently found among therapy-related AMLs, which may make it more relevant to the patient population treated at tertiary cancer centers. Methods: Using a retrospective, multimodal diagnostic approach, we present a cohort of patients with t(8;16) AML, describing the clinical history, cytomorphology, immunophenotype and cytogenetics associated with this unique subclass of AML. Results: 14 patients Median age 51, 50% of patients have extramedullary manifestations, most commonly leukemia cutis, which seemed to precede systemic involvement. Cytomorphology from 7 patients showed blasts...

Abstract 866 Background: Dramatic improvements have been made in treating children with newly diagnosed acute lymphoblastic leukemia (ALL). However, poor outcomes are still observed in patients who are either refractory to or who have relapsed after conventional chemotherapy. Salvage therapy options are urgently needed for this patient population. Clofarabine is approved as a single agent for treatment of pediatric ALL in second relapse. Previously we reported the safety profile and response rates from the phase 1 study assessing clofarabine in combination with etoposide and cyclophosphamide in pediatric patients with refractory/relapsed acute leukemia (Hijiya, Leukemia, 2009). Here we report the phase 2 results. Methods: Patients (pts) aged 1–21 years with refractory/relapsed ALL were treated at the recommended phase 2 dose of clofarabine 40 mg/m2/day, cyclophosphamide 440 mg/m2/day, and etoposide 100 mg/m2/day. All 3 agents were given IV daily for 5 consecutive days in induction and 4 consecutive days in consolidation. Patients could receive up to 2 induction cycles, followed by consolidation (max 8 cycles including induction). The primary endpoint was overall remission rate (ORR: complete remission [CR] and CR without platelet recovery [CRp]). Secondary endpoints were safety and tolerability, rate of partial remission (PR), duration of remission (DOR), event-free survival (EFS), 4-month EFS, and overall survival (OS). Minimal residual disease (MRD) by flow cytometry was evaluated as an exploratory endpoint. Results: Phase 2 comprised 25 pts (median follow-up 10.7 weeks [wks]): 16 males and 9 females with a median age of 14.0 years. Twenty-one pts had pre-B cell ALL, 1 pt had T cell ALL and 3 pts had an unknown immunophenotype. Fourteen pts had 2 prior induction regimens, 7 pts had 3 prior induction regimens and 4 pts had 1 prior induction regimen. Fifteen pts (60%) were refractory to their immediately preceding regimen. Four pts had received a prior hematopoietic stem cell transplant (HSCT). The investigator-assessed ORR was 44%; 7 CR (28%) and 4 CRp (16%). Additionally, 3 pts (12%) achieved PR. Eight pts were evaluable for MRD after induction, 5 were MRD negative (defined as Conclusions: Combination treatment with clofarabine, etoposide and cyclophosphamide in pediatric pts with refractory or relapsed ALL resulted in an ORR of 44% and negative MRD in heavily treated pts. Ten pts including 7 of 11 responders proceeded to HSCT. The safety profile is acceptable in this relapsed/refractory population. Disclosures: Hijiya: Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Clofarabine (Clolar) is approved by the US FDA for the treatment of pediatric patients 1 to 21 years old with relapsed or refractory acute lymphoblastic leukemia after at least two prior regimens. This trial examines the use of clofarabine in combination with etoposide and cyclophosphamide. Paul:Genzyme: Employment, Equity Ownership. Borowitz:genzyme: Research Funding; becton-dickinson: Research Funding; Alexion: Consultancy; beckman-coulter: Research Funding. Isakoff:Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Silverman:Genzyme: Research Funding; Enzon : Honoraria, Membership on an entity's Board of Directors or advisory committees; EUSA: Honoraria, Membership on an entity's Board of Directors or advisory committees. Steinherz:Genzyme: Research Funding. Kadota:Genzyme: Employment, Equity Ownership. Pressey:Genzyme: Research Funding. Shen:Genzyme: Research Funding. Chu:Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Cooper:Genzyme: Research Funding. Jeha:Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Razzouk:Genzyme: Research Funding. Rytting:Genzyme: Research Funding. Barry:Genzyme: Employment, Equity Ownership. Carroll:Genzyme: Research Funding. Gaynon:Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 3546 Between August 1988 and December 2009, 40 pediatric patients (pts) received an allogeneic HSCT for the treatment of very high risk ALL in CR1. They were 22 males and 18 females, aged 0.8 –19.2 years (median 10.0). Thirty eight patients had very high risk features: high white cell count greater than 200,000/mm3 (N=7), hypodiploidy (N=6), including double hypodiploidy (N=3), high risk cytogenetics with t(9;22) (N=14) or Mixed Lineage Leukemia (MLL) (N=3), and induction failure (N=23). Induction failure was defined morphologically by a single marrow aspirate revealing greater than 35% blasts by day +7 of induction, or greater than 5% blasts by day +14 or day +28 of induction. The remaining two patients included one infant ALL with a WBC >100,000/mm3 and one patient with ALL, Evans Syndrome and a prior CNS hemorrhage and fungal infection. Twenty two patients had one VHR feature while 16 patients had two VHR features. Patients received marrow or peripheral blood stem cell transplants from HLA-matched siblings (N=14) or volunteer unrelated donors (N=21) or unrelated double cord blood transplants (N=5). Unrelated donors were HLA-matched for 12 pts, but mismatched for nine pts at one (N=6) or two Class I antigens (N=3). All cord blood donors were matched at 4 or 5/6 HLA-antigens. Twenty three patients received T-cell depleted grafts following cytoreduction with hyperfractionated total body irradiation (HF-TBI) 1,375-1,500 cGy, Thiotepa (Thio) 5 mg/Kg/day × 2 and Cyclophosphamide (CY) 60 mg/Kg/day × 2. Grafts were marrow T-cell depleted with soy bean agglutinin and e-rosetting for 15 patients or peripheral blood stem cells T-cell depleted by CD34 selection and E-rosetting for 8 patients. Twelve pts received unmodified grafts following cytoreduction with a TBI-CY based regimen (N=8) or chemotherapy regimen (N=4). Five patients received double cord blood grafts following HF-TBI 1,375 cGY, Fludarabine 25 mg/m2/day × 3 and CY 60 mg/Kg/day × 2. Graft-versus-host disease (GvHD) prophylaxis for the BMT recipients included cyclosporine (CSA) + methotrexate or steroids, and for the cord blood recipients, CSA and mycophenolate mofetil. With a median follow-up of 11.1 years (range 0.6–21.9 yrs), the 10-year overall survival and disease-free survival of the entire cohort were 78% and 70% respectively, and 80% and 72% respectively when excluding the accidental death. Two recipients of T-cell depleted grafts suffered a graft failure (one early and one late), both of whom received a secondary T-cell depleted graft from a (different) mismatched related donor, engrafted and are alive and well. Two recipients of unmodified marrow grafts relapsed 4.9, and 18.9 months post BMT, one of whom is still alive disease free following treatment with chemotherapy. One recipient of a double cord blood graft relapsed three months post transplant. The overall risk of relapse for the entire cohort was 15%. Six pts died; cause of death was: relapse (N=2), secondary malignancy (N=2), acute GvHD (N=1), and accidental death (head trauma) (N=1). Grade 2–4 GvHD was diagnosed in recipients of unmodified grafts (N=3), double cord blood graft (N=2) and T-cell depleted grafts (N=1). The OS and DFS for recipients of T-cell depleted transplants following TBI THIO CY were both 83% with a 0% relapse rate. In summary, the outcome of allogeneic HSCT for children with very high risk ALL in CR1 in our series appears to be superior to that reported with chemotherapy for patients with these very high risk features. Moreover, the use of hyperfractionated TBI, Thio and CY followed by T-cell depleted grafts from related or unrelated donors was associated with a very good outcome with no relapse and minimal GvHD. Disclosures: No relevant conflicts of interest to declare.

Abstract 2363 The development of tMDS/tAML is a complication that can occur following chemotherapy (CT) or radiation (RT) therapy. Therapeutic approaches for such patients (pts) have included both CT and/or HSCT, with overall successful outcome in 30–35% of pts. From October 1994 to September 2009, 22 pts with tMDS/tAML received an allogeneic HSCT at our institution. The median age at presentation of primary disease was 15 years (range 3 months–26 years). The median age at time of HSCT was 21 years (range 8–29 years). There were 11 males and 11 females. Therapy related MDS/AML developed following RT and/or CT in pts with cancer (n=19) or a non-malignant disease (n=3). The initial malignancies consisted of Osteosarcoma (OS) n=5, Neuroblastoma (NB) n=3, Ewing Sarcoma n=3, Rhabdomyosarcoma n=2, Desmoplastic Round Cell Tumor n=1, Malignant Fibrous Histiocytoma n=1, Hodgkin Lymphoma n=1, Non-Hodgkin Lymphoma n=1, and AML n=1. Five pts proceeded to HSCT without induction chemotherapy, at which time four pts had refractory anemia (RA) and one pt had AML. The other 17 pts were treated with chemotherapy prior to transplant; 14 received high dose cytarabine at 3g/m2/dose every 12 hours × 4 doses with 13 pts achieving morphologic remission or secondary RA. Conditioning regimens were selected according to previous therapies: three pts had a total body irradiation (TBI) containing regimen; 11 pts received busulfan (Bu) (0.8mg/kg/dose every 6 hours × 10 doses) melphalan (Mel) (70mg/m2/dose × 2 doses) fludarabine (Flu) (25mg/m2/dose × 5 doses) for cytoreduction; and eight pts received myeloablative regimens containing Bu and/or Mel and/or thiotepa with doses modified for organ toxicity. Sixteen pts received T-cell depleted (TCD) bone marrow (n=2) or peripheral blood stem cell (PBSC) (n=14) grafts (including 11 pts following Bu-Mel-Flu); four received unmodified bone marrow (n=3) or PBSC (n=1); and two received a double umbilical cord blood transplant (DUCBT). Donors included HLA-matched (n=9), or mismatched related donors (n=3), HLA-matched (n=4), or mismatched (n=4) unrelated donors or DUCBT (n=2). Disease status at time of HSCT was: morphologic and cytogenetic CR (n=12); RA with positive cytogenetics (n=6); and refractory disease (n=4). The median follow up is 5.9 years (2.2-15.7 years). The five-year overall survival (OS) and disease free survival (DFS) rates for the entire group are 56.6% with 12 pts alive without evidence of either primary disease or tMDS/tAML. The OS and DFS rate for the 11 pts who received the Bu-Mel-Flu cytoreduction with TCD grafts was 54.5%. Ten pts died. Cause of death was relapse of MDS/AML (n=5) or of primary disease (n=2), GVHD (n=2), and infection (n=1). Four pts developed grade II-IV acute graft versus host disease (GVHD). Two pts recovered and two pts died from infectious complications related to GVHD. One patient developed localized chronic GVHD which resolved. Variables associated with a favorable outcome included: disease status with DFS of 63% for pts in RA or CR at HSCT compared to 0% for pts with >5% residual blasts in the bone marrow (p=0.04); age with DFS of 70% for pts Our results suggest that the strategy of induction with high dose cytarabine-based chemotherapy followed by allogeneic stem cell transplantation improves the overall outcomes for pts with tMDS/tAML to those obtained in pts transplanted for primary MDS/AML. In addition, the use of T-cell depleted transplant may decrease the toxicity of transplantation in heavily pre-treated pts without increase in relapse rate. Disclosures: No relevant conflicts of interest to declare.

Chemotherapy and radiation treatment of the central nervous system may cause delayed neurotoxicity in children with acute lymphocytic leukemia. We evaluated 12 long-term survivors of childhood leukemia using [18F]fluorodeoxyglucose positron emission tomography, computed tomography scans, clinical neurological examinations, and neuropsychological tests. Regional cerebral metabolic rate for glucose (rCMRGlc) values for white matter were lower in the older long-term survivors (greater than 18 years old) treated with cranial radiation and intrathecal chemotherapy than in normal control subjects or survivors who had been treated with intrathecal chemotherapy alone. The ratio of white matter: cortex rCMRGlc values was lower than control values in the long-term survivors treated with cranial radiation and intrathecal chemotherapy, regardless of age, but not in those treated with intrathecal chemotherapy alone. By contrast, thalamic rCMRGlc values were lower than control values in older survivors regardless of treatment, and the ratio for thalamus:cortex rCMRGlc values was lower in all the treatment groups than in the control subjects. The highest rCMRGlc values were found in the youngest children, indicating an important effect of age on cerebral glucose metabolism. No neuropsychological deficits were identified in patients treated only with intrathecal chemotherapy; however, lower IQ scores were found in the long-term survivors who had been treated with cranial radiation and intrathecal chemotherapy. Treatment of the central nervous system with cranial radiation and intrathecal chemotherapy may cause prolonged alterations in white-matter and thalamic rCMRGlc, which may permit the identification and assessment of neurotoxicity in long-term survivors of acute lymphocytic leukemia by [18F]fluorodeoxyglucose positron emission tomography.

Dose and dose intensity are believed critical for attaining a maximal therapeutic effect in drug-responsive tumor systems. Childhood acute lymphoblastic leukemia may be an example of such a drug-responsive system as it is cured with current chemotherapy in a majority of cases. Between August 1981 and May 1983, the Childrens Cancer Study Group enrolled 209 children with ALL and unfavorable presenting features in CCG-193P, a trial based on the Berlin Frankfurt Munster 76/79 regimen. The cumulative delivered dose of each medication was recorded prospectively. Patients who completed the intensive portion of therapy in continuous complete remission were ranked by the percentage of protocol required drug delivered from the initiation of therapy to that date. No association was found between delivery of any single drug and subsequent disease-free survival. However, when patients were ranked by the sum of the percentages of protocol vincristine, l-asparaginase, and anthracycline delivered, children in the approximate middle and lower tertiles were 3 and 5 times more likely to have had a subsequent relapse than were those in the upper tertile (P = 0.025, test for trend). Delivery of the full protocol prescribed dose of these agents may have been critical, but corroboration is certainly needed. Only prospective trials can determine if children with acute lymphoblastic leukemia and unfavorable presenting features might benefit from greater use of vincristine, l-asparaginase, and/or anthracycline.

We have previously reported encouraging overall complete remission (CR) rates and an acceptable safety profile using the combination of clofarabine, cyclophosphamide and etoposide in relapsed or refractory childhood acute leukemia. Here, we report follow-up results of the phase I portion of the study and provide an update on phase II which is currently enrolling patients. Patients between 1 and 21 years of age with refractory or relapsed acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML) (Phase I portion only) were enrolled. A standard 3+3 design was followed to determine the maximally tolerated dose combination in phase I. Five dosing cohorts evaluated escalating doses of clofarabine 20–40 mg/m2/day, etoposide 75–100 mg/m2/day and cyclophosphamide 340–440 mg/m2/day. All 3 study drugs were administered via IV infusion daily for 5 consecutive days in induction and 4 consecutive days in consolidation. Patients received up to 2 induction cycles, followed by consolidation (up to a maximum of 8 cycles in total). Dose-limiting toxicities in the phase I portion have been previously reported. The recommended phase II doses were clofarabine 40 mg/m2/day, cyclophosphamide 440 mg/m2/day, and etoposide 100 mg/m2/day. The phase II portion of the study is a single-arm open-label study with a planned total enrollment of 33 patients. Twenty-five patients (ALL: 20 patients; AML: 5 patients) were enrolled in the 5 phase I dose cohorts. The median number of prior induction regimens was 2, 7 patients (5 ALL, 2 AML) were refractory to their immediately preceding regimen, and 4 patients (1 ALL, 3 AML) had a prior hematopoietic stem cell transplant (HSCT). Based on investigator’s assessment, data showed complete remission (CR) in 10 patients (9 ALL, 1 AML) and complete remission without platelet recovery (CRp) in 6 patients (2 ALL, 4 AML) for an overall response rate of 64% (ALL: 55%; AML: 100%). Of the 16 responders (CR + CRp), 9 patients proceeded to HSCT after treatment. The median duration of remission (censored at the last known date of follow-up regardless of alternative therapy) for the 16 responders was 18.2 weeks (range 6.6 to 61.4 weeks) (ALL: median 31.4 weeks, range 6.6 to 61.4 weeks; AML: median 15.6 weeks, range 7.3 to 48.9+ weeks), including 31.4 weeks for the 10 patients with CR and 15.3 weeks for the 6 patients with CRp. At the last known date of follow-up, 7 of the 16 responders were alive and 4 of these remained in CR (three of these had undergone post-therapy HSCT). One patient with ALL completed 8 cycles of therapy, with a duration of remission of 61.4 weeks. In phase II, 3 of the first 8 ALL patients enrolled achieved a response (1 CR, 2 CRp). However, 4 patients developed severe hepatotoxicity (3 veno-occlusive disease, 1 hyperbilirubinemia). The study was amended to exclude patients with prior HSCT, viral hepatitis and/or cirrhosis, or elevated conjugated bilirubin levels. Four additional patients have since been enrolled in the amended phase II portion of the study and no cases of severe hepatotoxicity have been observed to date. In summary, the combination of clofarabine, cyclophosphamide and etoposide induced durable remission in children with relapsed or refractory acute leukemia. The recommended phase II doses of clofarabine, cyclophosphamide, and etoposide were 40 mg/m2/day, 440 mg/m2/day, and 100 mg/m2/day, respectively, each given for 5 days in induction. The phase II portion of the study is now actively enrolling patients with ALL without a history of prior HSCT following development of hepatoxicity in 4 patients. An update of all patients enrolled in phase II will be presented at the meeting.

21032 Background: Epstein-Barr virus (EBV) asymptomatically establishes persistent infections in more than 90% of the adult population. However, due to effective immune control, only a minority of infected carriers develops spontaneous EBV-associated lymphomas. Since EBV nuclear antigen-1 (EBNA1) is the only protein expressed in all proliferating EBV infected cells we hypothesize that EBNA1 specific immune response is critical in preventing EBV-positive lymphomas. Methods: After informed consent, peripheral blood from healthy volunteers and lymphoma patients (prior to therapy- no evidence of cytopenia) were stimulated (ex vivo) with overlapping peptides covering the immunogenic EBNA1 (aa400–641) sequence. Frequency of EBNA1-specific T-cells were assessed by intracellular cytokine staining and flow cytometric proliferation assays. Cytokine pattern, surface marker phenotype and functional reactivity against EBV specific and control antigens were analyzed. Results: Patient and volunteer immune responses to control antigens and other viruses were assessed and statistically indistinguishable. EBNA1 specific CD4+ T cell responses were detected among 18 of 20 healthy carriers, and among 10 of 16 patients with EBV-negative lymphoma (relative to healthy volunteers p=0.145 via paired student T test). None of the patients with EBV-positive lymphomas (n=8) had a detectable EBNA1-specific CD4+ T-cell response (p+ T cell responses. Although patients with EBV-positive lymphoma have intact immune responses to common viruses and antigens, they selectively lack an EBNA1-specific CD4+ T cell response. An intact EBNA1 specific immune response among patients with EBV-negaitve lymphoma implies that lymphoma is not a cause of a selective immune deficiency. On the contrary, these findings suggest that EBNA1-specific CD4+ T cells are critical in the prevention of EBV mediated lymphomas, and a defect in EBNA1 specific immunity may leave EBV carriers suseptible to EBV-positive lymphomas. EBNA1- specific CD4+ T cell function may be a new target for therapies of EBV-associated malignancies. No significant financial relationships to disclose.

Background: Clofarabine is a second generation purine nucleoside analogue synthesized to improve the properties of its congeners, fludarabine and cladribine. Clofarabine inhibits DNA polymerase alpha and ribonucleotide reductase, leading to the depletion of intracellular deoxynucleotide triphosphate pools, disruption of mitochondrial function and apoptosis. Methods: Clofarabine was administered by IV infusion over 2 hrs for 5 days at 52 mg/m2/day and repeated every 2–6 weeks based on bone marrow response and recovery of normal hematopoiesis. An independent review panel confirmed all results. Results: The median age of the 103 children (61 with ALL and 42 with AML) was 12 years (range 1–22 yrs). ALL: The overall remission rate (CR+CRp) was 20%; 30% (18/61) of patients showed a response (7CR, 5CRp, 6PR). Responses were noted in 15 of 50 (30%) patients with B- lineage ALL, 2 of 6 (33%) with T-cell ALL. Responders received a median of 3 prior induction regimens; 50% (9/18) had prior HSCT and 50% (9/18) were refractory to the preceding induction regimen. Response rate in refractory patients was 26% (9/35). After clofarabine treatment, 10 patients proceeded to transplant (including 8 responders). 6 of 10 patients who received a transplant were alive at last follow up (survival range: 30.1+ – 145.1+ wks). Response duration in 6 patients with CR or CRp who did not receive a transplant ranged from 4.3 to 58.6 weeks; 2 patients maintained CR for 47.9 and 58.6 weeks after clofarabine therapy. Median overall survival for the patients who achieved at least a PR was 66.6 weeks compared to 12.9 weeks for all patients. AML: The response rate for AML patients was 26% (1 CRp, 10 PR). Responders had received a median of 2 prior induction regimens, 36% (4/11) had prior HSCT and 55% (6/11) patients were refractory to the preceding induction regimen. Response rate in refractory patients was 21% (6/28). One patient who had received 5 prior induction regimens achieved CRp. 13 (31%) patients (1CRp, 6PR, 3NE, 3TF) underwent HSCT after completing clofarabine therapy, 5 of whom were alive at last follow-up (survival range: 62.7+ – 160.1+ wks). Many patients proceeded to HSCT as soon as a donor was identified without waiting for the patient to go into remission, making remission difficult to assess. Median overall survival for patients who achieved at least a PR was 32.1 wks compared to 23.4 wks for all patients. Safety: The side effect profile was not unexpected in this heavily pretreated population. The ≥ gr 3 drug related AEs occurring in ≥10% patients included febrile neutropenia for ALL patients; febrile neutropenia, pyrexia, nausea, neutropenia, and hypotension for AML patients. Conclusion: Clofarabine shows significant activity in heavily pretreated children with multiply relapsed acute leukemia, allowing otherwise refractory patients to proceed to HSCT. Durable responses were seen in ALL patients with no donor options who continued therapy with single agent clofarabine. Combination studies incorporating clofarabine in pediatric patients with relapsed ALL and AML are ongoing.

In prior studies, children and adolescents with markedly elevated (ME) WBC≥200,000 had a poor outcome. We examined the outcome of these patients enrolled on CCG l961 (n=250) and compared them to other patients with WBC

6588 Background: Clofarabine, a next-generation nucleoside analogue, has shown clinical activity when used as a single agent in heavily pretreated patients with relapsed or refractory acute leukemias. The results of two Phase 2 multicenter, open-label studies with clofarabine in children with refractory or relapsed ALL or AML are presented here. Methods: Clofarabine was administered at 52 mg/m2 given by intravenous infusion over 2 hours daily over 5 consecutive days. Cycles were repeated every 2 to 6 weeks based on response and toxicity. Results: 103 patients (61 ALL, 42 AML) have been treated. Patients on the ALL trial had received a median of 3 prior regimens (range 2–6). Thirty percent had received prior transplant. As determined by independent review, preliminary data indicate overall response rates of 30% in ALL (7 CR, 5 CRp, and 6 PR). Median duration of CR/CRp is 29 weeks. On the AML trial, median age was 12 years (range 2–22). Median number of prior regimens was 2 (range 1–5). Forty three percent ...

Recent CCG and POG treatment stategies for patients with T-ALL are significantly different. POG treats T-ALL patients on a separate protocol from that used for B precursor ALL (B-ALL) while CCG treats T-ALL patients on the same protocols utilized for B-ALL patients with protocol assignment determined by the NCI risk classification. We recently reviewed the outcome of patients with T-ALL and high-risk features (age ≥ 10 years and/or initial WBC > 50,000/ul) treated on either the CCG-1961 or POG-9404 clinical trials. POG utilized an intensive anthracycline based multidrug regimen based on the DFCI treatment program and randomized patients at registration to receive or not receive additional high dose methotrexate(HDMTX) with leucovorin rescue. All patients received cranial irradiation (CRT). CCG therapy for high risk T-ALL was based on CCG modified BFM(S-BFM) and Augmented BFM(A-BFM). Patients with

Background: Clofarabine, a next generation nucleoside analogue, was well tolerated and demonstrated activity in adult and pediatric Phase I trials conducted in heavily pretreated leukemia patients. Multicenter Phase II studies in pediatric leukemia have completed accrual in the US and are reported here. Methods: Two Phase 2, multicenter, open-label studies were conducted with clofarabine in children with refractory or relapsed ALL or AML. Clofarabine was administered intravenously over 2 hours at 52 mg/m2/day for 5 consecutive days. Cycles were repeated every 2 to 6 weeks based on response and toxicity. Results: The studies enrolled 100 patients (60 ALL and 40 AML). Currently, data are available for 84 patients (49 ALL, 35 AML). Median age is 12 years (range 1 to 22 years) and median number of prior regimens is 3 (range 1 to 6). Thirty-nine percent had received prior bone marrow transplant (BMT). As determined by independent review, preliminary data indicate overall response rates of 31% in ALL (6 CR, 4 CRp, and 5 PR) and 26% in AML (1 CRp and 8 PR). Median duration of remission for ALL is 9.7 weeks (range 1.0 to 28.6) and for AML is 16.2 weeks (range 1.7 to 56.6+). Thirteen of 24 responding patients (54%) proceeded to BMT. Median survival was 42 weeks (range 7.0 to 63.1+) for responding ALL patients (CR+CRp+PR) and 39 weeks (range 7.7 to 93.6+) for responding AML patients (CRp+PR). Patients who failed treatment or were non-evaluable had shorter median survival; 7.4 weeks (range 0.9 to 40.1+) and 12.4 weeks (range 1.6 to 84.9+) for ALL and AML, respectively. Among the patients who were refractory to the last prior chemotherapy, 7/30 (23%) with ALL and 4/22 (18%) with AML achieved a response with clofarabine. Median duration of remission in these patients is 4.6 weeks (range 2.3 to 24.4+) for ALL and 20 weeks (range 1.7 to 56.6+) for AML. Most drug-related adverse events were transient including febrile neutropenia, diarrhea, nausea/vomiting, fever, skin rash, headache, elevation in liver enzymes and bilirubin, and infusion-related flushing and anxiety. Conclusions: Clofarabine is active as a single agent in pediatric ALL and AML that are refractory to intensive salvage regimens. The overall safety profile is similar to that reported in other pediatric salvage studies. Clofarabine in combination with standard chemotherapy is currently under investigation in children.

AYA Patients 16–21 years of age with ALL are treated on both pediatric and adult ALL protocols. Recent publications have suggested that AYA patients treated on pediatric protocols have a significantly better outcome than those treated on adult protocols. From November 1996 to May 2002, 262 AYA ALL patients were entered on the CCG 1961 protocol. Treatment was based on the following observations from the previous CCG 1882 Protocol (1989–1995). 1) For patients with rapid early response (< = 25% blasts in a day seven bone marrow aspirate-RER), in the context of CCG modified BFM therapy (S-BFM), pre-symptomatic central nervous system therapy with intensive intrathecal (IT) methotrexate (MTX) alone provided equivalent results to standard IT MTX and cranial radiation (CRT). 2) For patients with slow early response (> than 25 blasts on day 7-SER), the augmented BFM (A-BFM) regimen produced a significant improvement in outcome compared to S-BFM. A-BFM featured a significant increase in dose intensity for vincristine (VCR), L-asparaginase (L-ASP), and steroid, the use of intravenous MTX without rescue in conjunction with VCR and L-ASP in interim maintenance (IM), and a second IM and delayed intensification (DI) phase. RER patients on 1961 were randomized to one of four arms; S-BFM, S-BFM with a second standard IM and DI phase, A-BFM with only 1 IM/DI phase, and full A-BFM. SER patients were randomized to A-BFM or A-BFM with Idarubicin/Cytoxan pulses added to the 2 DI phases. One hundred ninety patients had < 50K WBC (73.4%) and 21% had T cell immunophenotype. Seven patients had a t (9/22) and three patients had a t (4/11). Five year EFS for AYA patients treated on CCG 1961 was 68% ± 3.7% and the five-year survival was 77% ± 3.2%. The majority of events were isolated bone marrow relapses. For RER AYA patients, five year EFS for patients treated on the augmented intensity arms (N=87) was 83.2% compared to 60.8% for patients treated on the standard intensity arms (N=76) (P = .10). Within the augmented intensity arms, there was no difference in EFS for patients randomized to one or two DI phases. There was no difference in EFS for the 2 SER treatment arms. 5 year EFS was 78% for the Idarubicin/Cytoxan arm and 74% for the ABFM arm. Five year EFS for AYA patients with < 50K WBC was 73.2% (N=190) versus 54% for AYA patients with > 50K WBC. Unlike younger patients, there was no difference in outcome for males and females. Conclusions: 1) AYA patients with ALL have a 68% 5 year EFS when treated with BFM based chemotherapy. 2) WBC < 50K predicts for a favorable outcome. 3) Early augmented intensity therapy appears to improve outcome for AYA ALL patients showing a rapid response to induction therapy.

We have described a severe combined immunodeficiency (SCID) mouse model that permits the subcutaneous growth of primary human acute leukemia blast cells into a measurable subcutaneous nodule which may be followed by the development of disseminated disease. Utilizing the SCID mouse model, we examined the ability of patient-derived leukemic blasts to generate leukemic growth in the animals after subcutaneous inoculation without conditioning treatment. Leukemia blasts derived from 133 patients with acute leukemias, (67 acute lymphoblastic leukemia (ALL) and 66 acute myeloid leukemia (AML)), were inoculated into SCID mice. The leukemias displayed three distinct growth patterns: "aggressive", "indolent", or "no tumor growth". Out of 133 patients, leukemia samples 46 (34.5%) displayed an aggressive growth pattern, 14 (10.5%) indolent growth and 73 (55.0%) did not grow in SCID mice. The growth probability of leukemias from relapsed and/or refractory disease was 3 fold higher than that from patients with newly diagnosed disease. Serial observations found that leukemic blasts from the same individual, which did not initiate tumor growth at initial presentation and/or at early relapse, may engraft and grow in the later stages of disease, suggesting that the ability of leukemia cells for engraftment and proliferation was gradually acquired following the process of leukemia progression. Nine autonomous growing leukemia cell lines were established in vitro. These displayed an aggressive proliferation pattern, suggesting a possible correlation between the capacity of human leukemia cells for autonomous proliferation in vitro and an aggressive growth potential in SCID mice. It was demonstrated that patients whose leukemic blasts displayed an aggressive growth and dissemination pattern in SClD mice had a poor clinical outcome in patients with ALL as well as AML. Patients whose leukemic blasts grew indolently or whose leukemia cells failed to induce growth had a significantly longer DFS and more favorable clinical course.

8504 Background: Despite marked improvement in pediatric leukemia outcome, children with refractory disease or early relapse have a dismal prognosis with the current salvage regimens. Leukemia remains the leading cause of disease-related death in children. New therapeutic options are needed. Clofarabine, a next-generation nucleoside analogue, has shown clinical activity when used as a single agent in heavily pretreated children with relapsed or refractory acute leukemias. Methods: Two Phase 2 multicenter, open-label studies are being conducted with clofarabine in children with refractory or relapsed ALL or AML. Clofarabine is administered at 52 mg/m2/day over 5 consecutive days. Cycles are repeated every 2 to 6 weeks based on response and toxicity. Results: To date 69 patients (40 ALL, 29 AML) have been treated. Median age is 12 years (range 1 to 22 years) and median prior regimens is 3 (range 1 to 6). Forty-one percent had received prior transplant. As determined by independent review, preliminary data i...

Objective. —To assess the cardiac status of long-term survivors of pediatric malignancies who received chemotherapy, including anthracyclines. Design and Method. —Patients were evaluated by echocardiogram from 4 to 20 years (median, 7 years) after completion of anthracyclines, with prospective and retrospective analysis. Patients. —The consecutive sample of 201 patients had received a total anthracycline dose of 200 to 1275 mg/m2(median, 450 mg/m2), and 51 patients had mediastinal radiotherapy. Main Outcome Measures. —The overall incidence and severity of abnormal systolic cardiac function were determined for the entire cohort. Risk factors of total anthracycline dose, mediastinal radiotherapy, age during treatment, and length of follow-up were examined. Results. —Twenty-three percent (47/201) of the cohort had abnormal cardiac function on noninvasive testing at long-term follow-up. Correlation between total cumulative dose, length of follow-up, and mediastinal irradiation with incidence of abnormalities was significant. Fifty-six patients were followed up for 10 years or more (median, 12 years), with a median anthracycline dose of 495 mg/m2, Thirty-eight percent (21/56) of these patients, compared with 18% (26/145) of patients evaluated after less than 10 years, had abnormal findings. Sixty-three percent of patients followed up for 10 years or more after receiving 500 mg/m2or more of anthracyclines had abnormal findings. Nine of 201 patients had late symptoms, including cardiac failure and dysrhythmia, and three patients died suddenly. Microscopic examination of the myocardium on biopsy and autopsy revealed fibrosis. Conclusion. —The 23% incidence of late cardiac abnormalities warrants continued evaluation of patients after anthracyclines to guide patient care and the design of future chemotherapeutic protocols. (JAMA. 1991;266:1672-1677)

The prophylaxis of the CNS in ALL with intraventricular methotrexate therapy via an Ommaya reservoir in 18 patients resulted in a reduction of the CNS relapse rate to 11% with a 6–10+ year follow-up. This procedure proved to be safe and effective in our hands, but with newer, equally effective and less invasive methods of CNS prophylaxis now available, its use is no longer indicated. Intraventricular therapy via the Ommaya reservoir was also evaluated for its role in improving CNS remission duration after a leukemic recurrence in the CSF. The treatment of CNS relapse with intraventricular chemotherapy and low dose (600r) neuraxis radiation is encouraging. Of the ten patients treated for CNS leukemia, three are long-term (8–11+ years) survivors. Isolated CNS relapse occurred in three. The median CNS disease-free survival and the median relapse-free survival are 39 months and 21 months respectively.

The activity of terminal deoxynucleotidyl transferase (Tdt) was measured in mononuclear cells of 70 children with acute leukemia, in whom raised levels of Tdt were noted at diagnosis, relapse, or during remission. Serial measurements of Tdt activity were related to the percentage of blasts in the bone marrow and the clinical course of children both during therapy and after its completion. The Tdt values did not predict relapse in children with acute lymphoblastic leukemia nor did the quantitative determination correlate with the percentage of blasts in the bone marrow.

Data were collected from 124 patients with newly diagnosed acute lymphoblastic leukemia (ALL) and WBC greater than 200,000/microL seen at institutions affiliated with the Children's Cancer Study Group (CCSG) from April 1981 to May 1983. The presenting characteristics, initial management, early complications, and outcome were reviewed. All the children received vigorous intravenous (IV) hydration, alkalinization of the urine, and allopurinol. Thirty-two patients were started on full therapy with no additional measure. One or more special measures believed to reduce the complications of leukostasis and blast cell lysis were administered to 92 patients as follows: small initial doses of prednisone, 63; emergency cranial irradiation, 26; exchange transfusion, 21; and leukopheresis, 19. The incidence of CNS hemorrhage was only 3% (4/124). Seven patients expired during induction and four failed to achieve a remission by day 28. Nineteen patients (15%) had documented bacterial or fungal sepsis. Mild to moderate electrolyte abnormalities occurred in 29 patients: three patients required renal dialysis. Pretreatment with small doses of prednisone did not decrease the incidence of electrolyte abnormalities in those patients when compared with patients who received full chemotherapy. The event-free survival (EFS) for the 106 patients treated on one of the three intensive pilot studies is 55% at 36 months. On multivariate analysis the two significant adverse prognostic factors were massive splenomegaly (P = .02) and WBC count greater than 600,000/microL (P = .05). In conclusion, in patients with hyperleukocytosis the complications of blast cell lysis and leukostasis were manageable with acceptable morbidity and minimal mortality in a group of patients treated with vigorous hydration, allopurinol, and alkalinization of the urine before beginning chemotherapy. Selected patients with severe hyperuricemia and renal dysfunction may benefit from leukopheresis. No beneficial role was demonstrated for the use of small initial doses of prednisone or emergency cranial irradiation.

Splenectomy for massive splenomegaly in thrombocytopenic patients refractory to platelet transfusions carries increased surgical risks. Blocking of the splenic artery may reduce the size of the organ, prolong the survival of transfused platelets, and reduce the surgical complications. We describe four cases of extreme splenomegaly and thrombocytopenia where successful splenectomy was carried out after angiographic embolization of the splenic artery in children with ju venile chronic myelogenous leukemia. Significant improvement was observed in platelet counts and in the survival of platelets after transfusions in three of the patients. There was a concomitant decrease in transfusion requirements. Isoimmunization prevented prolonged platelet survival in the fourth case. We recommend earlier splenectomy in these patients to reduce transfusion requirements and delay the onset of poor platelet survival after transfusions.

The immunoregulatory activity of bone marrow leukemic blasts from five patients with null-cell acute lymphoblastic leukemia (ALL) and two children with T-cell ALL was evaluated. Blasts were studied in co-culture for their effects on the proliferative responses of normal allogeneic peripheral blood lymphocytes to phytohemagglutinin. Mitomycin C-treated null cell ALL blasts from two of five children suppressed the proliferative responses of normal responder cells by 80% and 58% whereas those from two other children enhanced the responses by 118% and 31%. T-cell leukemic blasts from one patient with T-cell ALL exhibited helper cell activity (26%) and T-leukemic blasts from the other demonstrated suppressor cell activity (53%). The helper cell activity of leukemic blasts was associated with stimulating capacity of null blasts in one-way mixed lymphocyte reactions. In six of seven cases, incubation of blast cells with concanavalin A (20 microgram/ml) for 48 hours prior to co-culture with responder cells did not result in the generation of significant suppressor cell activity. Our study suggests that leukemic blasts from certain patients with 'null' and T-cell ALL may possess spontaneous helper or suppressor cell immunoregulatory activity. This functional heterogeneity of leukemic blasts may help in subclassifying the ALL.

The authors treated 30 patients ages 1 to 19 with relapsed or refractory acute non-lymphoblastic leukemia (ANLL) with the combination of diaziquone (AZQ) and 2,2'-anhydro-arabinosyl-5-fluorocytosine (AAFC) intravenously in two dose schedules. The first 12 patients were treated with 19 courses of AZQ 30 mg/m2 X 3 days and AAFC 600 mg/m2/dose every 12 hours X 10 doses. An additional patient was treated with three courses at 80% of the above doses. Hepatic toxicity was National Cancer Institute Grade III in eight of 22 and Grade IV in one of 22 courses. Infectious complications were severe in all patients, including responders. One patient achieved a complete remission and one a partial remission; the latter died with marrow aplasia after a second course. Altogether three patients developed profound aplasia and died before marrow recovery. The authors treated a second group of 17 patients with AZQ 22.5 mg/m2/day X 3 days and AAFC 450 mg/m2/dose every 12 hours X 10 doses. Three patients achieved a complete remission and one patient a partial remission for 3, 4, 9, and 9 months, respectively. The remainder had progressive disease or no response. All patients developed profound myelosuppression but no toxic deaths occurred. Hepatic toxicity was reduced. Therapy induced cytoreduction of bone marrow was determined by flow cytometry in ten patients; none of these patients responded during the interval studied. Although AZQ and AAFC are active in childhood ANLL with acceptable toxicity, the combination does not appear more active than AZQ used alone. Future studies should define the role of AZQ in combination with other agents.

An intensive multimodal therapy was developed for the treatment of a subpopulation of children with acute lymphoblastic leukemia (ALL) who had a predicted event-free survival of less than 40% on previously reported therapeutic regimens (at high risk for early relapse). Induction with multiagent chemotherapy and radiotherapy to bulky disease-bearing areas (peripheral lymph nodes and mediastinum) was followed by consolidation, CNS prophylaxis, and cyclical remission maintenance therapy. Ninety-six (96%) of 100 previously untreated patients, 1 to 17 years of age, attained a complete remission. Seven patients received other maintenance therapy or a bone marrow transplant in remission. Sixty-six of the remaining 89 (74%) are in continuous complete remission at 22+ to 72+ months (median, 44+ months). Marrow relapse occurred in 15 (17%), CNS relapse in 5 (6%), and testicular relapse in one. Sixty-six of the 93 evaluable patients (71%) (including the induction failures) are event-free survivors. Two patients died of infection during the induction phase. No patient died during consolidation or maintenance without recurrent disease. The patients spent a median of 19, 0, and 0 days hospitalized during induction, consolidation, and maintenance, respectively. The most common complications were bacteremia and mucositis during induction and mucositis and fever during periods of neutropenia in consolidation. Maintenance was well tolerated. We conclude that the treatment protocol is intensive, but the inherent toxicities are manageable with adequate supportive care. The life table--projected event-free survival of 69% +/- 5% 48 months from diagnosis is encouraging.

Chemotherapeutic regimens for childhood acute lymphoblastic leukemia (ALL) include a remission induction period with high, daily doses of prednisone among other agents. A period of central nervous system (CNS) prophylaxis follows, during which steroids are often tapered entirely before cranial radiation (CRT) is completed or even initiated. The somnolence syndrome (SS) has been described 4 to 6 weeks after completion of CRT in up to 60% of the children with doses as low as 1800 cGy. A pilot study of continuous steroid coverage during CRT in childhood ALL was conducted. From July 1984 to July 1986, 38 children entered on Children's Cancer Study Group ALL protocols received CRT of 1800 cGy (180 cGy x 10). All patients received oral prednisone throughout the entire course of CRT at daily doses varying from 3.0 to 60.0 mg/m2. The overall incidence of the SS was 13% (five patients). The development of the syndrome was steroid dose-dependent: greater than or equal to 15 mg/m2/d (one of 32 patients), 3% incidence; less than 15 mg/m2 (four of six patients), 67% incidence. The presence of headache during CRT was also steroid dose-related: greater than or equal to 15 mg/m2, one of 32 patients; less than 15 mg/m2, six of six patients. Of the seven patients with headache during CRT, five developed the SS. The two patients (both of the less than 15 mg/m2 group) who did not develop the SS were the only cases treated with increased steroid doses at the onset of headache symptoms. Steroid coverage at a dose of greater than or equal to 15 mg/m2 during CRT appears to significantly reduce the incidence of acute radiation reactions and the SS. A prospective randomized study is planned to confirm these initial findings.

A 25 year old patient with congenital hypoplastic anemia (Black-fan-Diamond syndrome) is described. This patient was hepatitis-antigen negative, had not received androgens and had a hepatoma develop in a transfusional hemochromatotic liver. Since androgens have been associated with hepatocellular carcinoma, the use of androgenic steroids for other than life-threatening symptoms in this disease should be avoided.In this case report, the patient had been delivered by Caesarean section and weighed only 4 pounds at birth. The mother was O negative, the father A positive, and the infant A positive. Initial red cell count was 2.85 million/cu mm; white cell count, 19,200/cu mm; and hemoglobin 70% of normal. At 3 months of age hemoglobin was 10% of normal. Bone marrow examination revealed marked erythroid hyperplasia. A diagnosis of Blackfan-Diamond syndrome was made. He received blood transfusions every 2 or 3 weeks for the first 4 years of his life. During his lifetime he received 433 units of packed cells for the treatment of congenital hypoplastic anemia. Vitamin-B12, folic acid, and iron were given without benefit. At 8 years of age a spelectomy was done. 20 months after surgery he recovered from pneumonococcal meningitis without sequelae. Progressive signs of hemochromatosis developed and finally progressive signs of heart failure with edema. At 24 years of age severe epigastric pain developed. An open liver biopsy disclosed multiple liver nodules which proved to be hepatoma. Severe ascites followed the surgery. Pulmonary metastases of the liver tumor developed and heart failure. He died at age 25. This patient had received no androgen. He was consistently hepatitis antigen negative. He was prepubertal at the age of 25 and had almost no endogenous androgens. Alpha-fetoglobin was present. This test may be useful as a screening test for hepatoma.

During the National Influenza Immunization Program in 1976, 147 children with neoplastic diseases received Wyeth split-product bivalent influenza vaccine: A/New Jersey/8/76 (HSW1N1), A/Victoria/3/75 (H3N2). Thirteen normal siblings served as controls. Seventy-one patients received two doses of the vaccine four weeks apart. After the second injection of A/NJ/8/76, there was a difference between the response of the patients on chemotherapy and those off therapy greater than or equal to 30 days--38% vs. 76%, P less than 0.01 for four-fold rise and 26% vs. 57%, P less than 0.05 for the attainment of protective (greater than or equal to 32) hemagglutination inhibition (HI) titers. These differences were observed in both leukemia-lymphoma and solid tumor patients. There was a difference in HI titers to A/Vic/75 between patients on and off chemotherapy after a single injection, 34% vs. 71%, P less than 0.001 for a four-fold rise. After the second immunization, only 52% on, and 86% off therapy (P less than 0.05) had a four-fold rise in titers. Thirty-two percent of the patients on treatment who achieved "protective" titers did so only after the second immunization. Immunoglobulin levels and neutropenia did not correlate with the inability to obtain a four-fold rise in titers. Our findings suggest that patients on chemotherapy cannot be effectively vaccinated by a new antigen, and that single yearly boosters may be insufficient for recall of old antigens. Patients off chemotherapy greater than or equal to 30 days respond as normal controls.

Cerebrospinal fluid of 24 patients with acute leukemia was studied by DNA/RNA flow cytometry. In six of 15 patients with central nervous system (CNS) relapse, the spinal fluid cells had abnormal DNA stemlines, ranging from near haploid to hyperdiploid. In two additional cases, leukemic cells were identified by a abnormally high RNA content only. One patient had two different aneuploid cell populations in spinal fluid not distinguished by cytologic morphology. Another patient with initial diploid leukemia had CNS relapse characterized by the same DNA stemline long after a triploid DNA stemline emerged in the marrow. DNA/RNA flow cytometry detected leukemic cells that were not identified ("uniform" or "reactive") by cytological criteria in 5/6 patients studied and in addition differentiated lymphoblastic from nonlymphoblastic cell types according to low and high RNA content.

The Children's Cancer Study Group's (CCG) clinical trials in acute lymphoblastic leukemia (ALL) prior to 1981 consistently demonstrated that patients presenting with a white blood cell count (WBC) greater than or equal to 50,000/microliter or the "lymphoma syndrome" had a less than 40% 3-year event-free survival (EFS). The Berlin Frankfurt Munster (BFM) 76/79 study suggested that the prognosis of these patients could be improved. Before testing this therapy in a randomized setting, 29 CCG institutions used it for treatment of 209 newly diagnosed children with ALL and an initial WBC greater than or equal to 50,000/microliter or the lymphoma syndrome. In the intensive phases of therapy, 77% of cumulative parenteral doses and 55% of cumulative oral doses were within 10% of protocol requirements or were modified appropriately for reported toxicity. One hundred ninety-five patients achieved remission (93.3%). Eleven patients died in remission (5.6%)--10 during the intensive reinduction/reconsolidation phase. The 4-year EFS (+/- 1 SD) was 62% (+/- 3.7%) with a median follow-up of 40 months. Only one patient has had an isolated CNS relapse. These results appear superior to past CCG studies for high-risk patients and extend observations made from studies of similar therapy.

Thirty-four children after multiple relapse or with refractory acute lymphoblastic leukemia were treated with two novel combinations of high-dose cytosine arabinoside, methotrexate, asparaginase, vincristine, and prednisone. The first combination was given to 19 patients. Oncolytic response and marrow hypoplasia was achieved in all. There were four early infectious deaths. Thirteen of the remaining 15 (87%) in whom the response to therapy could be evaluated achieved complete remission. Two achieved good partial remissions. The median duration of complete remission and survival on study was 8 and 10 months, respectively. The four proven and three suspected fungal infections seen in the initial 19 patients was the major toxicity observed. The therapy was modified in the last 15 patients to retain efficacy while reducing the period of neutropenia from a median of 28 to 22 days. No deep-seated fungal infections were seen in these patients. Twelve (80%) achieved a complete remission. Three had an oncolytic response without achieving remission. Eighty-three percent of the 30 evaluable patients, or 74% of all patients entered on study, achieved remission. It is anticipated that the therapy described here will not only achieve another remission in the majority of patients with advanced ALL but that the patients will be able to proceed to alternate therapies with potentially more durable benefit.

Over the last 20 years, the rate of long-term disease-free survival of childhood acute lymphoblastic leukemia increased from less than 1 to 60 per cent. Strategies for disease control include (1) intensive multiagent induction to rapidly decrease the tumor burden and minimize the chance of emergence of a resistant population of cells, (2) restoration of normal hematopoiesis as soon as possible to reduce the period of pancytopenia, (3) meticulous supportive care, (4) prevention of sanctuary disease, and (5) therapy tailored to the potential risk of relapse as predicted by the prognostic factors of the patient. Further progress must be made in identifying prognostic factors, preventing and treating relapse, finding novel modalities of therapy, and delineating and minimizing long-term side effects.

Acute myeloblastic leukemia is suspected usually because of abnormalities of the peripheral blood or bone marrow. In some cases there may also be an extramedullary focus of leukemic cells called a chloroma or granulocytic sarcoma. On occasion, these tumor masses may precede any evidence of leukemia by months or years. They are often misdiagnosed as lymphomas or granulomas. We present here a case of acute myeloblastic leukemia that was preceded by a granulocytic sarcoma of the clavicle. The initial biopsy of the clavicle was thought to be consistent with Ewing's sarcoma. The patient was treated with systemic chemotherapy prior to gross marrow involvement. He has been in continuous complete remission 34 months after the start of chemotherapy and 44 months after the development of granulocytic sarcoma.

A patient with nodular sclerosing Hodgkin's disease and basal cell carcinoma syndrome is reported. Medulloblastoma and ovarian fibromas have already been associated with the syndrome and its coexistence with other malignant neoplasms has been described. The authors report here the first case of Hodgkin's disease with the multiple nevoid basal cell carcinoma syndrome. The fact that the patient's mother also has the syndrome, and the maternal grandfather had Hodgkin's disease seems to indicate more than a chance association of two independent events.