A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase.

Administration of chemotherapy is often limited by myelosuppression. Expression of drug-resistance genes in hematopoietic cells has been proposed as a means to decrease the toxicity of cytotoxic agents. In this pilot study, we utilized a retroviral vector expressing methylguanine DNA methyltransferase (MGMT) to transduce hematopoietic progenitors, which were subsequently used in the setting of alkylator therapy (procarbazine, CCNU, vincristine (PCV)) for poor prognosis brain tumors. Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells were collected by apheresis and enriched for CD34+ expression. Nine subjects were infused with CD34+-enriched cells treated in a transduction procedure involving a 4-day exposure to cytokines with vector exposure on days 3 and 4. No major adverse event was related to the gene therapy procedure. Importantly, the engraftment kinetics of the treated product was similar to unmanipulated peripheral blood stem cells, suggesting that the ex vivo manipulation did not significantly reduce engrafting progenitor cell function. Gene-transduced cells were detected in all subjects. Although the level and duration was limited, patients receiving cells transduced using fibronectin 'preloaded' with virus supernatant appeared to show improved in vivo marking frequency. These findings demonstrate the feasibility and safety of utilizing MGMT-transduced CD34+ peripheral blood progenitor cells in the setting of chemotherapy.

Mobilization and collection of peripheral blood CD34+ cells from patients with Fanconi anemia.

A potential therapeutic option for patients with Fanconi anemia is collection of peripheral blood stem cells prior to the development of severe pancytopenia. These hematopoietic cells potentially could be infused when symptomatic bone marrow failure develops, as autologous rescue after chemotherapy in the event of leukemic transformation, or as targets for gene therapy. Eight patients with Fanconi anemia were mobilized with 10 microg/kg per day of granulocyte colony-stimulating factor (median, 10 +/- 4 days) to determine the feasibility of collecting peripheral blood stem cells for future use. Six patients achieved a peripheral blood CD34+ count of > or = 6/microL and underwent apheresis. The collection goal was 2 x 10(6) CD34+ cells/kg based on a predicted weight 5 years from the date of collection. A mean of 2.6 +/- 0.9 x 10(6) CD34+ cells/kg of the weight at the time of collection were collected, which corresponded to 1.9 +/- 0.4 x 10(6) CD34+ cells/kg of the target weight. The collections required a mean of 4 +/- 3 days (range, 2-8 days) of apheresis. Six of the 8 subjects had > or = 1 x 10(6) CD34+ cells/kg cryopreserved based on both actual and target weights, and 4 subjects had > or = 2 x 10(6) CD34+ cells/kg cryopreserved based on the target weight. These results suggest that some patients with Fanconi anemia can have adequate numbers of CD34+ cells mobilized and collected from the peripheral blood prior to the onset of severe bone marrow failure, but they may require an extended mobilization and multiple days of collection.

A randomized comparison between rasburicase and allopurinol in children with lymphoma or leukemia at high risk for tumor lysis.

Standard therapy in the United States for malignancy-associated hyperuricemia consists of hydration, alkalinization, and allopurinol. Urate oxidase catalyzes the enzymatic oxidation of uric acid to a 5 times increased urine soluble product, allantoin. Rasburicase is a new recombinant form of urate oxidase available for clinical evaluation. This multicenter randomized trial compared allopurinol to rasburicase in pediatric patients with leukemia or lymphoma at high risk for tumor lysis. Patients received the assigned uric acid-lowering agent for 5 to 7 days during induction chemotherapy. The primary efficacy end point was to compare the area under the serial plasma uric acid concentration curves during the first 96 hours of therapy (AUC(0-96)). Fifty-two patients were randomized at 6 sites. In an intent-to-treat analysis, the mean uric acid AUC(0-96) was 128 +/- 70 mg/dL.hour for the rasburicase group and 329 +/- 129 mg/dL.hour for the allopurinol group (P <.0001). The rasburicase versus allopurinol group experienced a 2.6-fold (95% CI: 2.0-3.4) less exposure to uric acid. Four hours after the first dose, patients randomized to rasburicase compared to allopurinol achieved an 86% versus 12% reduction (P <.0001) of initial plasma uric acid levels. No antirasburicase antibodies were detected at day 14. This randomized study demonstrated more rapid control and lower levels of plasma uric acid in patients at high risk for tumor lysis who received rasburicase compared to allopurinol. For pediatric patients with advanced stage lymphoma or high tumor burden leukemia, rasburicase is a safe and effective alternative to allopurinol during initial chemotherapy.

Large-scale mobilization and isolation of CD34+ cells from normal donors.

We describe collection and purification of peripheral blood CD34+ cells from volunteer, normal donors and allogeneic stem cell donors. A total of 98 aphereses were performed on 68 volunteer donors using peripheral venous access. The mean number of nucleated cells collected was 4.6 x 10(10) which included 1.9 x 10(8) CD34+ cells corresponding to 2.7 x 10(6) CD34+ cells/kg. The number of CD34+ cells collected did not differ between males and females but did correlate with the donor's weight and the total number of nucleated cells collected. The Nexell Isolex 300i cell separator was used to isolate CD34+ cells from 30 of the collections. A mean of 0.36% of the total cells was recovered and included 43 +/- 18% of the CD34+ cells. CD34+ cells represented 85 +/- 11% of the recovered cells. The total number of CD34+ cells recovered was not influenced by the number of nucleated cells placed on the Isolex 300i. The percentage of CD34+ cells recovered was not related to the number of CD34+ cells placed on the Isolex 300i. The purity of the final product was influenced by the number of CD34+ cells but not the total number of nucleated cells. An additional 38 CD34+ cell isolations were performed on normal allogeneic stem cell donors with similar results. These observations further support the safety and feasibility of peripheral blood CD34+ cell collection and purification.

Prognostic significance of early response to a single dose of asparaginase in childhood acute lymphoblastic leukemia.

PURPOSE: The in vitro and in vivo efficacy of a single dose of asparaginase in children with newly diagnosed acute lymphoblastic leukemia and the correlation between in vitro and in vivo antileukemic response and long-term outcome were prospectively evaluated. PATIENTS AND METHODS: Two hundred fifty-one patients were randomized to receive 1 of 3 asparaginase preparations (Escherichia coli, Erwinia chrysanthemi [Erwinia], or pegaspargase). In vitro assessment of efficacy was expressed as the percent total cell kill (TCK), based on the number of viable cells found after 5 days of culture in the presence of asparaginase. In vivo leukemia cell kill (LCK) was calculated by comparing bone marrow cellularity and percent leukemic blasts in marrow obtained before and 5 days after treatment with a single dose of asparaginase. Acute toxicity was determined by clinical and laboratory assessment. RESULTS: There was equivalent cell kill with all three types of asparaginase. The mean in vitro TCKs for E. coli, Erwinia, and pegaspargase were 31%, 39%, and 36%, respectively (P = 0.63). The mean LCKs in marrow of patients exposed to E. coli, Erwinia, and pegaspargase were 69%, 74%, and 65%, respectively (P = 0.88). The lack of response to asparaginase in vitro predicted a higher risk for clinical relapse regardless of risk assignment (12 leukemic events among 21 in vitro nonresponders; 57%, P < 0.001). There was no difference in acute toxicity among the three asparaginase preparations. CONCLUSIONS: All three asparaginase preparations produced equivalent LCKs in in vitro and in vivo analyses. In vitro response to asparaginase provided a risk group-independent prognostic factor.

High dose chemotherapy with autologous peripheral blood progenitor cell transplantation in an anephric child with multiply recurrent Wilms tumor.

PURPOSE: An autologous peripheral blood progenitor cell (APBPC) transplant in an anephric child with multiply recurrent Wilms tumor using a conditioning regimen of high dose chemotherapy in conjunction with hemodialysis (HD) and peritoneal dialysis is described. PATIENT AND METHODS: The child had a left nephrectomy at 9 months of age for a stage II Wilms tumor. At 6 years of age, she required a right nephrectomy because of progressive, recurrent disease unresponsive to treatment with doxorubicin, actinomycin, and vincristine. She was maintained on peritoneal dialysis. Salvage chemotherapy consisted of 5 cycles of carboplatin and cyclophosphamide after APBPCs were collected after granulocyte colony-stimulating factor mobilization. After a preparative regimen of carboplatin, cyclophosphamide, and etoposide with closely timed HD, peripheral blood progenitor cells were infused and peritoneal dialysis was resumed. RESULTS: No nonhematopoietic toxicity occurred. Pharmacokinetic studies demonstrated that HD effectively eliminated carboplatin and provided safe, effective plasma concentrations in this anephric patient. Trilineage engraftment occurred by day +10 and the child was discharged from the hospital on day +14. She had a local recurrence on day +194 and died of progressive disease on day +660. CONCLUSIONS: With dialysis support and dose modification, high-dose chemotherapy followed by APBPC transplantation can be successfully performed in the anephric child. Given the lack of organ toxicity in this patient, increased doses of the drugs used in this preparative regimen may be possible for anephric children.

High dose cyclophosphamide with carboplatin: a tolerable regimen suitable for dose intensification in children with solid tumors.

PURPOSE: To determine the hematopoietic and nonhematopoietic toxicity of a novel dose-intensive chemotherapy regimen for the treatment of children with relapsed solid tumors. PATIENTS AND METHODS: The time to hematopoietic recovery and toxicity experienced during 46 courses of high-dose cyclophosphamide (4.0 g/m2), MESNA, and carboplatin (400 mg/m2) with granulocyte colony stimulating factor (G-CSF) support in 14 children with recurrent solid tumors was reviewed. RESULTS: All patients developed grade 4 neutropenia and thrombocytopenia. Recovery to an absolute neutrophil count (ANC) of 500/microliter and platelet count of 50,000/microliter occurred at a median of 15 days and 23 days respectively. Median time to ANC > 1,000/microliter and platelets > 100,000/microliter was 27 days. Hospitalization for fever and neutropenia occurred during 35 of 46 courses, with documented bacteremia in six courses. There was no grade II or greater nonhematopoietic organ toxicity. Responses (CR + PR) were observed in 6 of 11 evaluable patients. CONCLUSIONS: These data suggest that this regimen is tolerable in heavily pretreated children with solid tumors with myelosuppression as the primary toxicity. Due to the lack of significant nonhematopoietic toxicity, this is a good candidate regimen for dose escalation using peripheral blood progenitor cell infusions and deserves further evaluation for efficacy in children with both recurrent and newly diagnosed high-risk solid tumors.

Coming to terms: parents' response to a first cancer recurrence in their child.

The purpose of this study was to explore, using grounded theory, the process experienced by parents who are dealing with the first recurrence of cancer in their child. The sample of 33 guardians (27 mothers, 1 grandmother, and 5 fathers) was drawn from three pediatric oncology settings. Data were collected through interviews, observations, and medical record review. Thirteen parents were interviewed to validate first the evolving and, later, the complete study findings. Four interactive components emerged: regulating shock, situation monitoring, alternating realizations, and eyeing care-limiting decisions. The overall organizing construct induced from these components was labeled "coming to terms." This construct represents the parents' efforts to overcome shock and despair to make wise decisions about treatment while accepting that the outcome if beyond their control, and to help their child have the optimal chance for cure while preparing for the child's possible death.

Predicting the risk of bacteremia in childen with fever and neutropenia.

PURPOSE: We sought to identify factors assessable at the time of admission for fever and neutropenia that predict bacteremia in children with cancer. PATIENTS AND METHODS: One hundred fifteen consecutive episodes of fever and absolute neutrophil count (ANC) less than 500/microliter in 72 children with cancer were studied prospectively to determine the risk of bacteremia using data assessable at the time of presentation. After exploratory analysis identified admission temperature and absolute monocyte count (AMoC) as the strongest predictive factors, recursive partitioning was used to determine cutpoints for these variables that resulted in discrimination between episodes associated with a lower or higher risk of bacteremia. RESULTS: There were 24 episodes of bacteremia (21% of episodes). Episodes were grouped using the cutpoints for AMoC and temperature: 17% were classified as low risk for bacteremia (AMoC > or = 100/microliter), 65% as intermediate risk (AMoC < 100/microliter and temperature < 39.0 degrees C), and 18% as high risk (AMoC < 100/microliter and temperature > or = 39.0 degrees C). No episodes classified as low risk were associated with bacteremia; 19% of intermediate-risk and 48% of high-risk episodes were associated with bacteremia. The odds ratio of bacteremia for the high-risk versus the intermediate-risk group is 4.4 (95% confidence interval, 1.6 to 12.9). The risk classification was validated using data from 57 different episodes of fever and neutropenia treated in the same hospital. CONCLUSION: Three levels of risk for bacteremia are defined using the AMoC and temperature at the time of admission for fever and neutropenia. Trials now should be conducted to test whether these factors may be used to assign some children to less intensive or outpatient antibiotic therapy at the time of presentation with fever and neutropenia.

Molecular genetics: toward an understanding of childhood cancer.

Investigation in cellular and molecular genetics has yielded a wealth of new information regarding the role of genetic alterations in the etiology of childhood cancer. The recent advancement of molecular biologic techniques has allowed researchers to bridge the gap from the description of chromosomal aberrations in tumor cells to the current identification of oncogenes and tumor suppressor genes involved in malignant transformation. This article reviews the advances in molecular genetics of childhood cancer, briefly outlines the history, and details the current knowledge of the use of cytogenetics, oncogenes, and other tools in the diagnosis, management, and further understanding of pediatric malignancies.

Stage III neuroblastoma over 1 year of age at diagnosis: improved survival with intensive multimodality therapy including multiple alkylating agents.

PURPOSE: A nonrandomized, single-arm trial was conducted to assess the efficacy of multimodality therapy including intensive chemotherapy with multiple alkylating agents in the treatment of children with Evans stage III neuroblastoma older than 1 year at diagnosis. PATIENTS AND METHODS: Twenty-five patients with a median age of 18 months at diagnosis were treated with multimodality therapy including surgery and chemotherapy using either nitrogen mustard (mechlorethamine), doxorubicin, cisplatin, dacarbazine (DTIC), vincristine, and cyclophosphamide (MADDOC) or cisplatin and cyclophosphamide induction followed by maintenance MADDOC (induction MADDOC) protocols. Sixteen of 25 patients also received radiotherapy to the tumor bed and primary lymph nodes. Event-free survival (EFS) was compared with that reported previously in the literature. N-myc amplification was evaluated prospectively and the Shimada classification was evaluated retrospectively as potential prognostic factors. RESULTS: We report a 72% EFS (95% confidence interval +/- 18%) with a median follow-up of 85 months. EFS was significantly worse for patients with tumors demonstrating N-myc amplification (P = .018). Patients classified as favorable according to the Shimada system experienced a significantly better EFS (P = .04), but unfavorable patients still maintained a 60% EFS. CONCLUSION: Intensive multimodality treatment including MADDOC and induction MADDOC chemotherapy provides a very good EFS for children older than 1 year who have stage III neuroblastoma. Children classified as favorable according to the Shimada system have a better prognosis. Patients whose tumors demonstrate N-myc amplification have a poor prognosis despite therapy.

Incidence of secondary acute myelogenous leukemia after treatment of childhood acute lymphoblastic leukemia.

BACKGROUND: Recent reports of secondary acute myelogenous leukemia (AML) occurring in children previously treated for acute lymphoblastic leukemia (ALL) prompted a review of patients with ALL treated at the Dana Farber Cancer Institute consortium (DFCI) between 1973 and 1987. Seven hundred fifty-two of 779 children treated for ALL entered complete remission. The mean follow-up time for the 752 patients was 4.4 years. Two children had AML develop 12 and 13 months after the diagnosis of ALL, respectively. METHODS: The estimated overall risk of secondary AML was calculated for the patient population as instances per 1000 patient-years of follow-up. This was compared with recent reported cases from another institution. RESULTS: The estimated overall risk of secondary AML was 0.61 instances per 1000 patient-years of follow-up (95% confidence interval: 0.15, 4.4). The difference between the risk of 0.61 among DFCI patients versus previously reported risk of 5.8 among a differently treated group of patients with ALL was statistically significant (P = 0.0008). No epipodophyllotoxin was used in the patients in the DFCI consortium. In contrast, an epipodophyllotoxin was used in 12 of 13 previously reported patients who had secondary AML develop. CONCLUSIONS: The authors concluded that the use of epipodophyllotoxins may be associated with an increased risk of having secondary AML develop in patients with ALL.

Positive and negative elements regulate human interleukin 3 expression.

The human interleukin 3 (IL-3) promoter is comprised of several cis-acting DNA sequences that modulate T-cell expression of IL-3. These are located within 315 nucleotides upstream of the mRNA start site. Transient expression of reporter genes linked to serially deleted sequences of the IL-3 promoter has allowed mapping of two activator sequences and an interposed repressor sequence. The proximal regulatory region is specific to IL-3 and prerequisite for efficient transcription. Its effect is enhanced by a second, more distal activating sequence consisting of an AP-1 binding site. Between the two activators lies a transcriptional silencer, which is a potent repressor in the absence of the AP-1 site. DNA-nuclear protein binding experiments demonstrate specific complex formation within each of these functional regions. Thus, both positive and negative regulatory elements appear to control expression of the human IL-3 gene in activated T cells.

The isolation of ecdysterone inducible genes by hybridization subtraction chromatography.

We have developed a procedure for selectively enriching a mRNA population for inducible sequences. Other than the induced mRNA species, the population of mRNA in control cells is approximately the same as the mRNA population in induced cells. Cytoplasmic mRNA from control cells is bound to oligo (dT)-cellulose and used as a template for reverse transcriptase, the oligo (dT) serving as a primer. After removing the template mRNAs, the cDNA-cellulose column is used to hybridize a population of mRNAs from induced cells. The non-hybridized poly A+ RNAs are greatly enriched in the inducible sequences. We have used this technique of hybridization subtraction chromatography to select a mRNA population enriched for the mRNAs inducible by ecdysterone in Schneider's Line 2 Drosophila cells. This population of RNAs was used to screen a recombinant library. Preliminary results indicate that approximately 10% of the RNA in the probe population represents ecdysterone inducible sequences. Methods are described for optimizing the cDNA synthesis reaction (we obtain greater than or equal to 30% efficiency) and hybridizing RNA to the cDNA-cellulose resin. This method can be used to select induced mRNAs regardless of the way in which the induction is brought about.