CancerTherapy: Clinical

A Phase I Clinical, Pharmacologic, and Biologic Study of Thrombopoietin and Granulocyte Colony-Stimulating Factor in Children Receiving Ifosfamide, Carboplatin, and Etoposide for Recurrent or Refractory Solid Tumors: A Children’s Oncology Group Experience Anne L. Angiolillo,1Virginia Davenport,2 Mary Ann Bonilla,2 Carmella van deVen,2 Janet Ayello,2 Olga Militano,2 Langdon L. Miller,3 Mark Krailo,4 Gregory Reaman,4 and Mitchell S. Cairo2

Abstract Purpose: Ifosfamide, carboplatin, and etoposide (ICE) are associated with grade III/IV dose- limiting .The Children’s Oncology Group conducted a phase I dose escalation, pharmacokinetic, and biological study of recombinant human thrombopoietin (rhTPO) after ICE in children with recurrent/refractory solid tumors (CCG-09717) to assess the toxicity and maximum tolerated dose of rhTPO administered at1.2,2.4, or 3.6 Ag/kg per dose. Experimental Design: Children received ifosfamide 1,800 mg/m2 on days 0 to 4, carboplatin 400 mg/m2 on days 0 to 1, and etoposide 100 mg/m2 on days 0 to 4. rhTPO was administered i.v. on days +4, +6, +8, +10, and +12at1.2,2.4, or 3.6 Ag/kg per dose. Results: rhTPO was well tolerated and maximum tolerated dose was not reached. Median time to recovery z100,000/AL of rhTPO at 1.2, 2.4, and 3.6 Ag/kg/d was 24 days (22-24d), 25 days (23-29d), and 22 days (16-37d), respectively. Patients required a median of 2 days of plate- let transfusions (0-7 days). Mean (F SD) rhTPO maximum serum concentrations were 63.3 F 9.7 and 89.3 F 15.7 ng/mL and terminal half-lives were 47 F 13 and 6 4 F 42 hours after 2.4 and 3.6 Ag/kg/d, respectively.There was a significant increase in colony-forming unit up- on WBC count recovery. Conclusions: rhTPO was well tolerated. Time to hematologic recovery and median number of platelet transfusions seem to be improved compared with historical controls receiving ICE + gran- ulocyte colony-stimulating factor (CCG-0894).

Approximately one third of children with malignant solid with an overall response rate (complete response + partial tumors experience either refractory or recurrent disease requir- response) of 51% (1). Grade III/IV thrombocytopenia, however, ing retrieval therapy. We have previously shown that ifosfamide, remains the major dose-limiting toxicity (DLT) for dose carboplatin, and etoposide (ICE) combination chemotherapy is intensification of ICE chemotherapy in these children (1). an excellent retrieval regimen in these patients and is associated Children with recurrent/refractory solid tumors and receiving ICE + granulocyte colony-stimulating factor (G-CSF) had a 92% incidence of grade III/IV thrombocytopenia and required a median of six platelet transfusions during the first cycle of ICE Authors’ Affiliations: 1Children’s National Medical Center, George Washington chemotherapy; only 30% of patients recovered their platelet University, Washington, District of Columbia; 2Childrens Hospital of New York count by day 21 to start cycle 2 (1). Presbyterian, Columbia University, New York, New York; 3Pharmacia Corp., Regulation of megakaryocytopoiesis is controlled in part by 4 Peapack, New Jersey; and Keck School of Medicine, University of Southern several hematopoietic including (IL)-1, California and Children’s Oncology Group, Arcadia, California IL-3, IL-6, IL-11, granulocyte-macrophage colony-stimulating Received 9/23/04; revised 12/28/04; accepted 1/6/05. Grant support: Division of Cancer Treatment, National Cancer Institute, NIH, factor, , fibroblast , , Department of Health and Human Services, and Pharmacia Corp. inhibitory factor, and thrombopoietin (2, 3). Throm- The costs of publication of this article were defrayed in part by the payment of page bopoietin, the most potent that regulates platelet charges. This article must therefore be hereby marked advertisement in accordance production, is a 60- to 70-kDa polypeptide of 322 amino acids with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Presented in part at the Annual Meeting of the American Society of Clinical (4–7). Thrombopoietin has both megakaryocyte colony- Oncology, June 2003, Chicago, Illinois. stimulating activity and maturation activity (4) and is encoded Contributing Children’s Oncology Group investigators, institutions, and grant by a single-copy on human 3q27-28 (7). numbers are given in the appendix. Thrombopoietin is produced primarily in the and Requests for reprints: Anne L. Angiolillo, Children’s Oncology Group, PO Box and is expressed in muscle stromal cells, bone marrow, and 60012, Arcadia, CA 91066-6012. Phone: 626-241-1509; Fax 626-445-4334; E-mail: [email protected]. spleen (8, 9). We (M.S.C.) have previously showed an inverse F 2005 American Association for Cancer Research. relationship of circulating thrombopoietin serum levels and

Clin Cancer Res 2005;11(7) April 1, 20 05 2644 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Thrombopoietin in Children Receiving ICE circulating platelet counts in patients with thrombocytopenia therapy and received no colony-stimulating factors for 10 days. Patients after ablative stem cell transplantation (10). who had received craniospinal irradiation (>3,600 cGy) or radiation Studies in animal models have showed the safety and efficacy therapy (including total body irradiation) to >50% of bone marrow of thrombopoietin to reduce thrombocytopenia associated with space were ineligible. Patients who had received the exact combination and dosage of ICE within the last 3 months were also ineligible. The chemo- and radiotherapy (7, 11). Using normal and myelosup- patient or legal guardian must have signed a documented informed pressed mice (carboplatin/irradiation regimen), Kaushansky consent approved by the institutional review board indicating et al. showed that thrombopoietin administration was associ- awareness of the investigational nature and risks of this study. ated with statistically significant increases in marrow and spleen Chemotherapy administration. Patients received ifosfamide 1,800 erythroid, granulocyte-macrophages, and megakaryocytic pro- mg/m2 on days 0 to 4, carboplatin 400 mg/m2 on days 0 to 1, and genitor cells (12). Platelet and red cell recovery was also etoposide 100 mg/m2 on days 0 to 4. 2-Mercaptoethane sulfonic acid accelerated in thrombopoietin-treated myelosuppressed mice and i.v. hydration were administered during each of the 5 days of compared with controls. Likewise, Grossman et al. have shown ifosfamide. Chemotherapy was repeated every 21 days and the absolute that treatment with thrombopoietin accelerates recovery of neutrophil count (ANC) was z1,000/AL and platelet count was z platelet, red blood cell, and neutrophil counts and improves 100,000/AL. G-CSF was discontinued at least 2 days before subsequent their nadirs in myelosuppressed mice (carboplatin plus total chemotherapy. Chemotherapy dose modifications for renal impairment and hematuria were protocol directed. There was no dose modification body irradiation; ref. 13). Furthermore, administering throm- for bone marrow suppression. Surgical tumor resection and radiation bopoietin to cynomolgus monkeys with chemotherapy- therapy for palliation and/or to obtain a partial or complete response of induced (nimustine) thrombocytopenia reduced the severity of local lesions was to be considered after the patient had completed two thrombocytopenia and accelerated the platelet recovery rate in a courses of chemotherapy and response to ICE was assessed. dose-dependent fashion (14). Most importantly, we (M.S.C.) Colony-stimulating factor administration. RhTPO was provided and have shown in a carboplatin-myelosuppressed murine model distributed by Pharmacia & Upjohn, Inc. (Peapack, NJ), in 3-mL vials that thrombopoietin after carboplatin effectively improved containing 2 mL of 0.1 mg/mL rhTPO in buffer solution. All patients platelet nadir and platelet recovery (15). received rhTPO according to an assigned cohort level. Three dose levels Clinical trials in humans have shown thrombopoietin to were used: 1.2, 2.4, and 3.6 Ag/kg per dose. Patients received rhTPO i.v. enhance platelet recovery in adult patients with cancer on days +4, +6, +8, +10, and +12 (five doses total). There was no rhTPO dose modification in this study. (recombinant human met after myelosuppressive chemotherapy and/or radiotherapy G-CSF) was injected s.c. at 5 Ag/kg/d for all rhTPO dose levels (16–18). In addition, in a phase I/II clinical trial, 12 adults beginning 24 hours after chemotherapy ended. G-CSF was continued with sarcoma received a single i.v. dose of thrombopoietin until the postnadir ANC was z1,000/AL for 2 consecutive days, or 28 without chemotherapy. Platelet counts increased markedly in days maximum. RhTPO could be discontinued if a patient developed a dose-dependent manner and no major side effects were documented DLT (excluding hematologic) related to rhTPO, evidence observed (16). Platelet response was accompanied by a dose- of grade III/IV thrombosis, thrombocytopenia thought to be related related increase in bone marrow colony-forming unit (CFU) to neutralizing rhTPO antibody formation, or platelet count and expansion and mobilization of myeloid, z1,000,000/AL after platelet nadir. erythroid, and megakaryocytic progenitor cells (16). Vadhan-Raj Recombinant human thrombopoietin dose escalation. The rhTPO et al. recently reported the results of a clinical trial in adults with dose level was assigned at enrollment and there was no intrapatient dose sarcoma and showed the importance of timing recombinant escalation. DLT was defined as any grade IV nonhematologic toxicity that was definitely, probably, or possibly related to rhTPO. Dose human thrombopoietin (rhTPO) administration before and escalation was not considered until at least three evaluable patients had after chemotherapy for optimal effect on platelet nadir and been entered at the current dose level. If none experienced DLT, the dose recovery (19). was escalated to the next higher level in the three subsequent patients. If The Children’s Oncology Group conducted a study (CCG- one of the three patients experienced DLT at the current dose, up to three 09717) of rhTPO plus G-CSF in children receiving ICE more were accrued at the same level. If none of these three additional chemotherapy for recurrent or refractory solid tumors. This patients experienced DLT, then the dose was escalated in subsequent dose-escalation phase I trial investigated pharmacokinetics and patients. If one or more of those three additional patients experienced toxicities associated with administering rhTPO after ICE. DLT, the maximum tolerated dose (MTD) would be deemed exceeded Additional aims included evaluating the depth and duration and three more patients would be treated at the next lower dose (unless of neutropenia and thrombocytopenia, evaluating the number six patients had already been treated at that prior dose). The MTD was the dose level at which fewer than one third of patients of days of platelet transfusion events during ICE chemotherapy experienced DLT. No intrasubject dose escalation was permitted. Only with rhTPO plus G-CSF, and quantifying peripheral blood CFU the toxicity evaluation made during the first course of therapy was used megakaryocyte before and after ICE with rhTPO plus G-CSF. to determine MTD. Hematologic recovery. Platelet recovery was defined as when an Materials and Methods unsupported platelet count remained above 100,000/AL for 1 day in the absence of a prior platelet transfusion within the previous Patient eligibility. CCG-09717 was opened for patient accrual in 72 hours. Complete blood count (CBC), differential, and platelet November 1998 and was closed in November 2002. Patients with counts were obtained on days 0, 4, 6, 7, then every other day on days recurrent or refractory solid tumors who were >1 year and V21 years of 8 to 18 and day 21, and daily when either the platelet count was age were eligible for study entry. All patients (excluding those with <20,000/AL, ANC <500/AL, ANC >10,000/AL, or platelet count brain stem tumor) were required to have histologic verification of >100,000/AL during each course. malignancy and radiological or histologic evidence of recurrence at Recombinant human thrombopoietin pharmacokinetics. Blood sam- initial diagnosis. Disease categories were sarcoma (soft tissue and bone) ples were drawn for pharmacokinetic analysis during the first course of and kidney, brain, and other solid tumors (gonadal, germ cell, therapy for patients receiving 2.4 and 3.6 Ag/kg rhTPO doses only. malignant melanoma, retinoblastoma, liver, and miscellaneous tumor). Serum rhTPO concentrations were assessed at the following time Patients must have recovered fully from toxic effects of any prior points: day 4—baseline predose, 10 minutes, then 3, 6, 12, and

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washing away unbound substances, a second enzyme-linked monoclo- Ta b l e 1. Patient characteristics (evaluable patient) nal antibody specific for bound thrombopoietin was added to the wells. After a wash to remove unbound antibody enzyme reagent, a substrate Medianageatstudyentry(y) 6.5(2-17) solution was added to the wells and color developed in proportion to the amount of thrombopoietin bound in the initial step. The color Sex, M/F 5/7 development was stopped and the intensity of the color was measured. Diagnosis RhTPO concentrations were determined using a calibration curve Osteosarcoma 3 with a working range of 31.2 to 2,000 pg/mL. The between-run Primitive neuroectodermal tumor 2 precision was 5.9% coefficient of variation, and the analytic recovery Hepatoblastoma 2 ranged from 83% to 103% for rhTPO added to serum. Serum samples Ewings 1 for rhTPO antibodies were screened by ELISA based on full-length Glioma 1 thrombopoietin, C-mpl receptors, or both. Reactive sera were tested Fibrosarcoma 1 using a bioassay based on inhibition of the thrombopoietin-dependent Rhabdomyosarcoma 1 cell line. Neutralizing antibodies were defined as those that were Desmoplastic small cell 1 inhibitory on bioassay and associated with clinically significant thrombocytopenia. Dose levels (Ag/kg) For all subjects, average serum rhTPO concentration-time curves were Dose level 1 (1.2) 3 plotted for 2.4 and 3.6 Ag/kg dose groups. Across subjects and doses, Dose level 2 (2.4) 3 maximum serum rhTPO concentrations and areas under the concentra- Dose level 3 (3.6 Ag/kg) 6 tion-time curve for day +4 doses were examined for dose proportion-

ality. In addition, maximum (10 minutes, Cmax) and minimum (predose, Cmin) serum rhTPO concentrations for days +6, +8, +10, 24 hours postdose; days 6, 8, and 10—5 minutes before and 10 minutes and +12 were summarized by dose group and examined across the after rhTPO administration; day 12—predose, 10 minutes, then 24, 48, dosing period to assess accumulation. A two-compartment open model and 72 hours postdose. Patients also were evaluated for presence of with i.v. bolus input and first-order output was fitted to serum rhTPO anti-rhTPO antibodies on day 5 of each course until removed from data obtained after the two dose levels using weighted (l/Yobs) nonlinear protocol therapy. An additional antibody level was obtained if platelet regression analysis (WinNonlin, Version 1.5, Scientific Consulting, Inc., count did not recover by day +35. Blood samples were collected into Apex, North Carolina). Choice of the model and of the weighting plain redtop tubes and allowed to clot at room temperature. The function was done based on the residual analysis and of the coefficients samples were then centrifuged and the separated serum stored frozen of variation of the estimated parameters. Pharmacokinetic parameters between 20jC and 70jC. were compared at all dose levels. Incidence of anti-rhTPO antibodies RhTPO serum concentrations were determined using a quantitative was tabulated and antibody titers were summarized by dose level and ELISA. The assay used a combination of two monoclonal antibodies to time since day 1 of treatment phase. From the fitted equation, the thrombopoietin. The first was a capture antibody that was precoated to pharmacokinetic parameters were calculated as follows: area under the the wells of a microtiter plate and used to extract thrombopoietin from serum concentration versus time curve after the first dose (AUC0-1)= the samples. Standards and samples were pipetted into the wells and the Dose/Vc/K10, CL = Dose/ AUC0-1, Vss =CL MRT where CL is the immobilized antibody bound any thrombopoietin present. After serum clearance, Vss is the volume of distribution at steady state, and MRT is the mean residence time. Peripheral blood progenitor cells. Peripheral blood progenitor responses and subsets of progenitors were determined at the following time points. Peripheral blood was drawn at day 0 of course 1 before ICE chemotherapy and every other day until ANC is z1,000/AL after nadir. Briefly, for CFU megakaryocyte progenitor determination, -density, peripheral blood mononuclear cells 105/mL were suspended in Iscove’s media and added to collagen/media solution containing IL-3, IL-6, and rhTPO (StemCell Technologies, Vancouver, British Columbia, Canada). Cells were then dispensed to a chambered slide and incubated at 37jC,

5% CO2, and >95% humidity. After 10 to 12 days, slides were dehydrated, fixed, and stained with GPIIb/IIIa monoclonal antibody, and colonies counted. Statistical analysis. Any patient who completed the scheduled rhTPO administration was considered in the MTD of thrombopoie- tin. Patients were evaluated for time to hematologic recovery for each

Ta b l e 2 . Hematologic recovery during cycle 1 ICE chemo- therapy

rhTPO dose levels (Mg/kg) 1. 2 2 . 4 3 . 6 Platelet z100,000/AL 24 (22-24) 25 (23-29) 22 (16-37) ANC z1, 0 0 0 / AL 20 (19-24) 20 (18-21) 19.5 (17-33) Fig. 1. Probability of platelet recovery z10 0,0 00/AL after ICE chemotherapy. Probability of platelet recovery z10 0,00 0/AL as determined by the Kaplan-Meier method following rhTPO and G-CSF after ICE chemotherapy (all thrombopoietin NOTE: Values are median number and range of days (in parentheses). dose levels combined).

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Results

Patient characteristics. Between November 1998 and No- vember 2002, 16 patients, ages 1 through 18 years (median, 6.5 years) with recurrent or refractory solid tumors were enrolled in this dose-escalation phase I study (CCG-09717). Among the 16 patients enrolled, 12 were evaluable for toxicity (Table 1). Four patients were inevaluable because of progressive disease (n = 1), lack of administration of full-dose chemotherapy (n = 2), and surgery (n = 1). Median number (range) of courses completed for rhTPO dose level 1.2, 2.4, and 3.6 Ag/kg were 1 (1-2), 2 (1-5), and 2.5 (2-4), respectively. Hematologic recovery and platelet transfusions during cycle 1. The median number and range of days to platelet recovery z100,000/AL for rhTPO dose levels are illustrated in Table 2 and Fig. 1. Patients received a median of 2 days (0-7 day) of platelet transfusions. The median number and range of days to ANC recovery z1,000/AL for rhTPO dose levels 1.2, 2.4, and 3.6 Ag/kg are illustrated in Table 2 and Fig. 2. RhTPO pharmacokinetics. The pharmacokinetics of rhTPO Fig. 2. Probability of ANC recovery z1,000/AL after ICE chemotherapy. Probability of ANC recovery z1,0 0 0 / AL as determined by the Kaplan-Meier were studied in nine patients who received rhTPO at the method following rhTPO and G-CSF after ICE chemotherapy (all thrombopoietin 2.4 (n =3)and3.6Ag/kg dose (n = 6). Mean rhTPO dose levels combined). pharmacokinetic parameters are summarized in Table 3. Detectable serum concentrations of thrombopoietin were course of therapy delivered. Any patient who was evaluable for observed in all patients before the first rhTPO dose. The mean determination of the MTD and had submitted serial peripheral blood baseline thrombopoietin concentration was 1.22 ng/mL (range, counts was considered a candidate for this analysis. The time to ANC 0.3-2.2 ng/mL). Mean baseline thrombopoietin concentration recovery was calculated as the number of days from the start of the for each dosing level was 1.63 F 0.52 and 1.02 F 0.69 ng/mL for course to the first CBC in which the ANC exceeded 1,000/AL. Any patients receiving 2.4 and 3.6 Ag/kg, respectively (Table 3). CBC done in the interval from the start of chemotherapy to day 9 of Because these levels were very low, no correction was applied in the course was not considered because peripheral blood counts the analysis. Primary pharmacokinetic parameters following day dropped precipitously for all patients during this time frame. From 4 rhTPO administration are described in Table 4. Concentration among the remaining reported CBCs, the date the patient’s CBC was versus time curves after single and repeated rhTPO adminis- first above 1,000/AL (‘‘recovered date’’) was identified. Next, the date closest to the recovered date at which the patient’s ANC was trations are presented in Fig. 3. The high variability of the half- <1,000/AL (‘‘last below date’’) was identified. Patients removed from life (Table 3) after the 3.6-Ag/kg dose was due to a single patient therapy because of disease progression before recovery of ANC outlier with a terminal half-life of 149 hours. The volume of z1,000/AL were censored for recovery of ANC at the time of last distribution values at steady state were 66 F 9 and 94 F 34 mL/ reported CBC. Because of patterns of patient care, the last below and kg at 2.4 and 3.6 Ag/kg dose levels, respectively. The peak rhTPO recovered dates were separated by an average of 2 days (range, 1-7). concentrations were achieved 10 minutes after drug adminis- The survivor function for the recovery of ANC to at least 1,000/AL, tration at each dose level. A dose-dependent increase in mean therefore, was estimated using Turnbull’s interval censoring method- Cmax and AUC0-1 values was noted between two dose groups ology (20). A similar method was followed to estimate the time to (Table 3). Significant accumulation of rhTPO was not observed, recovery of to 100,000/AL. Results of the progenitor studies are expressed as mean values F SE. although modest increases in trough concentrations were noted The equality of the mean biological measures between baseline and with repeated dosing (Fig. 3). posttherapy time points was assessed using a Student’s t test. The fold Peripheral blood colony-forming unit megakaryocyte progenitor change was calculated by dividing the mean posttherapy value by the cells. Megakaryocyte progenitors (CFU megakaryocytes) were mean pretherapy value. evaluated at baseline and when the WBC had recovered to

Ta b l e 3 . Pharmacokinetic results following first-dose rhTPO in children after ICE chemotherapy

M Dose level ( g/kg) No. patients Cmin (ng/mL) Cmax (ng/mL) AUC0-111 (ng.h/mL) CL (mL/h/kg) Half-life (hour) 2.4 3 1.63 F 0.51 63.3 F 9.7 2,350 F 630 1.08 F 0.33 47.4 F 13.4 3.6 6 1.02 F 0.69 89.3 F 15.7 3,0 66 F 846 1.24 F 0.30 64.4 F 41.7

Abbreviations: Cmin, minimum concentration (baseline); Cmax, maximum mean concentration; AUC0-1, area under the concentration time curve extrapolated to infinity; CL, systemic clearance.

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Ta b l e 4 . Compartmental pharmacokinetic parameters of rhTPO after day 4 i.v. administration of 2.4- and 3.6-Ag/kg doses to children after ICE chemotherapy

1 1 1 Patient no. K10 (h ) K12 (h ) K21 (h ) Vc (mL/kg) Half-life (h) AUC0-111 (ng.h/mL) CL (mL/h/kg) Vss (mL/kg) Dose 2.4 Ag/kg 1 0.026 0.247 0.199 32.5 61.7 2,839 0.85 72.8 2 0.035 0.112 0.169 42.3 35.0 1,640 1.46 70.2 3 0.024 0.030 0.066 38.6 45.4 2,573 0.93 55.9 Mean (SD) 0.028 (0.006) 0.130 (0.110) 0.145 (0.070) 37.8 (4.9) 47.4 (13.4) 2,350 (630) 1.08 (0.33) 66.3 (9.1) Dose: 3.6 Ag/kg 4 0.032 0.193 0.194 38.2 45.0 2,944 1.22 76.2 5 0.032 0.549 0.440 25.6 49.0 4,344 0.83 57.5 6 0.028 0.176 0.216 51.6 46.2 2,483 1.45 93.6 7 0.035 0.039 0.038 43.3 52.3 2,397 1.50 88.6 8 0.041 0.178 0.124 37.1 44.6 2,360 1.53 90.6 9 0.019 0.034 0.016 50.3 149.2 3,867 0.93 156.6 Mean (SD) 0.031 (0.008) 0.195 (0.188) 0.171 (0.154) 41.0 (9.6) 64.4 (41.7) 3,066 (846) 1.24 (0.30) 93.8 (33.5)

Abbreviations: K10, elimination rate constant; K12 , transfer rate constant from central compartment1to compartment 2; K21, transfer rate constant from compartment 2 to compartment 1; Vc, volume of distribution to central compartment. z 1,000/AL after the nadir was determined and there was a Discussion significant (P < 0.001) increase of peripheral blood CFU megakaryocyte formation (Fig. 4). In an attempt to improve the incidence of grade III/IV Recombinant human thrombopoietin toxicity and antibody thrombocytopenia, reduce platelet transfusions and/or improve formation. There were no grade II to IV toxicities probably the percentage of patients recovering their platelet counts by or definitely related to rhTPO administration. Furthermore, day 21, we investigated the addition of IL-6 to ICE + G-CSF in there was no evidence of DLT attributable to rhTPO at any of this same population. We previously reported in this same the three rhTPO levels. There were no episodes of toxic-related journal that IL-6 was quite toxic and intolerable and, deaths related to rhTPO or ICE administration. Antibody furthermore, failed to improve the results of ICE + G-CSF formation to rhTPO did not occur in any patient. A without IL-6 (21). thrombopoietin MTD was not reached. This phase I trial evaluated the safety of rhTPO after ICE chemotherapy in pediatric patients with recurrent/refractory solid tumors. In this study, thrombopoietin was well tolerated in all patients and there was no evidence of DLT or antibody formation at any dose level. The MTD of rhTPO after ICE in children has yet to be determined. In the adult experience with thrombopoietin, thrombocytosis (platelet count z1 106/AL) and/or thrombosis has been seen. These toxicities, however, were not observed in the pediatric trial. We have conducted a series of four trials of ICE chemotherapy in children with recurrent/refractory solid tumors with different hematopoietic cytokine combinations in an attempt to enhance hematologic recovery and/or reduce the need for platelet transfusions (1). In CCG-0894, we administered identical doses of ICE and post-ICE G-CSF (5 versus 10 Ag/kg/d), resulting in a median of 21 (14-42) days to ANC recovery z1,000/AL, a median of 27 (15-65) days to platelet recovery z100,000/AL, and a median of 6 days of platelet transfusions (1). In comparison, in the present study, the median time to ANC recovery z1,000/AL in cycle 1 after ICE followed by rhTPO (3.6 Ag/kg) and G-CSF was 19.5 (17-33) days. Median time to platelet recovery z100,000/AL in cycle 1 was 22 (16-37) days. The median number of days of platelet transfusions during cycle 1 was 2 days (0-7). Hematologic recovery time for ANC, platelet counts, and number of transfusions required improved for those Fig. 3. Concentration (conc) versus time curve of rhTPO. Mean F SD serum concentrations of rhTPO after single and repeated i.v. administrations of 2.4- and patients receiving ICE followed by rhTPO plus G-CSF compared 3.6-Ag/kg doses to children after ICE chemotherapy. with ICE + G-CSF (1).

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at 2.4 Ag/kg, it seems that rhTPO has a higher T1/2 (47.4 F 13.4 hours), Cmax (63.3 F 9.7 ng/mL, and AUC (2,350 F 630 ng.h/mL) and is cleared less rapidly in children than adults. Thrombopoietin is synthesized at a constant rate and is metabolized and cleared by the binding to C-mpl receptor on platelets and megakaryocytes (10). The higher number of cells expressing C-mpl, the lower the thrombopoietin serum level. These results suggest that children with thrombocytope- nia receiving ICE for recurrent/refractory solid tumors may have fewer megakaryocytes or platelets expressing C-mpl than adults with thrombocytopenia after ablative therapy and stem cell transplantation (24). The increase in peripheral blood CFU megakaryocyte formation after myelosuppressive chemotherapy seen in our study is in agreement with results reported in several phase I studies with thrombopoietic growth factors. Murray et al. reported small increases in CFU megakaryocyte frequency and a >5-fold increase in CFU granulocyte, erythroid, monocyte, Fig. 4. CFU megakaryocyte responses in patients receiving rhTPO. Megakaryocyte megakaryocyte (CFU-GEMM) frequency in the peripheral progenitors were evaluated by placing light-density, peripheral blood mononuclear blood of adult sarcoma patients after administering rhTPO cells (105/mL) into collagen/media solution containing IL-3, IL-6, and rhTPO. After 10 to 12 days, slides were dehydrated, fixed, and stained with GPIIb/IIIa before chemotherapy (25). A phase I study of PIXY321 (IL-3/ monoclonal antibody, and colonies counted. Column, mean number of CFU granulocyte macrophage colony-stimulating factor fusion pro- megakaryocyte colonies before and after recovery of WBC z1,0 0 0 / AL (all thrombopoietin dose levels combined); bar, SE. tein) in adult sarcoma patients before chemotherapy reported a significant increase in CFU-GEMM (26). Similarly, we reported a significant increase in CFU-GEMM compared with baseline We recently studied ICE with IL-6 plus G-CSF (CCG-0931) with PIXY321 after ICE in children with recurrent/refractory in children with recurrent/refractory solid tumors, to deter- solid tumors (27). Further, CFU-GEMM colony formation mine safety, biological activity, and hematopoietic recovery significantly increased over baseline in our study of children effects (21). IL-6 is a pleiotropic cytokine that affects with refractory/recurrent solid tumors treated with ICE fol- megakaryocytopoiesis (22, 23). Nineteen patients were evalu- lowed by IL-6 with G-CSF (21). able for toxicity and received IL-6 at doses of 2.5 (n = 8), 3.75 In summary, the administration of rhTPO is well tolerated (n = 5), or 5.0 (n =6)Ag/kg/d. The MTD exceeded the lowest in children with recurrent/refractory solid tumors. No DLT or dose tested and DLTs were principally severe constitutional antibody formation was noted at any rhTPO dosing level and symptoms. All patients receiving IL-6 with G-CSF developed the MTD in children after myelosuppressive chemotherapy grade IV thrombocytopenia and required a median of five has yet to be determined. A randomized study would be platelet transfusions during course 1, which seems to be a required to determine if ICE + thrombopoietin + G-CSF higher number compared with our current thrombopoietin study (21). versus ICE + G-CSF in children with recurrent/refractory solid The pharmacokinetics of rhTPO administered i.v. postche- tumors significantly enhances time to platelet recovery and/or motherapy in children were characterized by low clearance reduces the need for platelet transfusions. and limited distribution into total body water. Wolff et al. showed, in an adult population receiving a similar rhTPO Acknowledgments dose (2.4 Ag/kg), Cmax (39.6 F 20.2 ng/mL), clearance (3.22 F F F 1.5 mL/h/kg), T1/2 (37.1 7.3 hours), and AUC0-1 (844 We thank Linda Rahl, Donna Correia, and Shaun Mason for their expert editorial 319 ng.h/mL; ref.24). Comparing these results with children assistance in the development of this article.

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Clin Cancer Res 2005;11(7) April 1, 20 05 2650 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. A Phase I Clinical, Pharmacologic, and Biologic Study of Thrombopoietin and Granulocyte Colony-Stimulating Factor in Children Receiving Ifosfamide, Carboplatin, and Etoposide Chemotherapy for Recurrent or Refractory Solid Tumors: A Children's Oncology Group Experience

Anne L. Angiolillo, Virginia Davenport, Mary Ann Bonilla, et al.

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