Thrombopoietin, the ligand for the c-Mpl receptor (found on platelets and megakaryocyte progenitors), was recently cloned by several investigators and was shown to be a primary regulator of platelet production in vivo [4-8]. Thrombopoietin promotes both the proliferation of megakaryocyte progenitors and their maturation into platelet-producing megakaryocytes. In preclinical studies done in normal mice and nonhuman primates, thrombopoietin increased platelet counts to a level higher than those previously achieved with other thrombopoietic cytokines [9, 10]. Moreover, in a murine model for myelosuppression, recombinant thrombopoietin given as a single dose decreased the nadir and accelerated platelet recovery in mice that had been rendered pancytopenic by sublethal radiation and chemotherapy [11]. In these studies, more prolonged treatment (for as long as 8 days) provided no additional benefit and was associated with marked thrombocytosis during the recovery phase.

On the basis of these observations, we initiated a phase I and II clinical and laboratory investigation of recombinant human thrombopoietin in patients with cancer who were at high risk for severe chemotherapy-induced thrombocytopenia. This trial was divided into two parts: Part I studied thrombopoietin given before chemotherapy, and part II studied thrombopoietin given after chemotherapy. The objective of part I, the results of which are reported here, was to assess the hematopoietic effects, pharmacodynamics, and clinical tolerance of this novel agent in patients who had normal hematopoietic function before chemotherapy.

Methods

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Patients

Patients with sarcoma who had never had chemotherapy, were suitable candidates for subsequent chemotherapy, and did not have rapidly progressive disease were eligible for this trial. Patients were required to have a Karnofsky performance status score of 80 or more, adequate bone marrow (absolute neutrophil count 1.5 x 10⁹/L; platelet count 150 x 10⁹/L and 450 x 10⁹/L), adequate renal function (serum creatinine level 120 µmol/L), and adequate hepatic function (alanine aminotransferase level < 3 times normal; bilirubin level < 1.5 times normal). Patients with a history of thromboembolic or bleeding disorders, significant cardiac disease, or previous pelvic radiation were excluded. Written informed consent was obtained from all patients before study entry in accordance with institutional guidelines.

Design

During the phase I dose-ranging portion of this clinical cohort study, thrombopoietin was administered as a single intravenous dose 3 weeks before chemotherapy. At study entry, three patients were assigned to each of four dose levels (0.3, 0.6, 1.2, and 2.4 µg/kg of body weight). Patients who had no dose-limiting toxicity and did not develop neutralizing antibodies to thrombopoietin were eligible to receive thrombopoietin at the same doses after chemotherapy.

Recombinant Human Thrombopoietin

The thrombopoietin used in this study was provided by Genentech, Inc. (South San Francisco, California). Thrombopoietin is a full-length glycosylated molecule produced in a genetically modified mammalian cell line and purified by standard techniques. It was mixed with preservative-free normal saline as a diluent for injections.

Clinical and Laboratory Monitoring

Before and during the clinical trial, patients were monitored by complete histories; physical examinations; and laboratory tests, including a complete blood cell count with differential counts, serum chemistry, coagulation profile, urinalysis, assessment of thrombopoietin antibody formation, chest radiography, and electrocardiography. Blood counts were obtained daily for the first 5 days and then at least three times per week. Peripheral smears were examined serially for platelet morphology. Platelet counts and the average size of platelets (mean platelet volume) were derived from 64-channel platelet histograms.

Bone marrow aspiration and biopsy were done before and 1 week after thrombopoietin treatment. The bone marrow specimens were initially fixed in 10% neutral formalin, embedded in paraffin, cut into sections 5 µm thick, and stained with hematoxylin-eosin for morphologic analysis and with Masson trichrome for analysis of collagen fiber content. Fresh, air-dried smears of bone marrow were stained with Wright-Giemsa. Bone marrow samples were examined for overall cellularity and morphology in a blinded manner. Megakaryocyte counts were measured by choosing 10 high-power (40x) fields in areas without artifactual zones or trabecula. The relative size of the megakaryocyte was assessed by examining bone marrow aspirate smears using the Magiscan Image Analysis System (Compix, Cranberry, Pennsylvania). Bone marrow aspirates were also assayed for hematopoietic progenitor cell number and cycle status, for content of CD34⁺ and CD41⁺ cell subsets (by flow cytometry), and for megakaryocyte ploidy (by flow cytometry). Blood samples were assayed for hematopoietic progenitor cell number and for platelet function.

Pharmacokinetics Profiles

Serum samples were collected before and at 2, 5, 10, 60, and 90 minutes and 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, and 120 hours after thrombopoietin administration. Concentration-time profile at each dose level was evaluated by using standard pharmacokinetics methods. Serum thrombopoietin levels were quantitated by enzyme-linked immunosorbent assay for thrombopoietin [12].

Hematopoietic Progenitor Cell Assays

Assays for colony-forming unit-granulocyte-macrophage (CFU-GM); burst-forming unit-erythroid (BFU-E); and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) using low-density bone marrow [13] and peripheral blood cells [14] were done with methyl cellulose assays. The percentage of bone marrow CFU-GM and BFU-E in DNA synthesis (S-phase of cell cycle) was measured by a high-specific-activity tritiated thymidine suicide technique [15]. Assays for colony-forming unit-megakaryocyte (CFU-MK) and burst-forming unit-megakaryocyte (BFU-MK) were done using a fibrin clot assay [16].

Ploidy Analysis

Megakaryocyte-enriched cell fractions were prepared from bone marrow cell suspensions by using a Percoll gradient technique. Ploidy was determined by flow cytometric measurement of the relative DNA content after staining with propidium iodide in hypotonic citrate solution [17]. Cells were also stained with anti-CD41b (8D9)-FITC (SyStemix, Palo Alto, California) to allow gating on CD41b⁺ megakaryocytes. At least 3000 CD41⁺ events were collected for each sample. The percentage of CD41⁺ cells in ploidy class was determined from the fluorescence-activated cell-sorting dot plots.

Platelet Function

Platelet aggregation was measured in response to three agonists: adenosine diphosphate (final concentration, 20 µg/mL), collagen (6 µg/mL), and thrombin (5 µg/mL). Standard methods were used [18]. The concentrations of agonists were chosen on the basis of previous in vitro studies done on blood from normal controls. The instruments used for the assays were the Bio/Data Pap 4A (Horsham, Pennsylvania) and the Crono-log 560CA (Havertown, Pennsylvania).

Immunophenotypic Analysis

Immunophenotypic analysis was done using anti-CD34 (Becton Dickinson, San Jose, California) and anti-CD41 monoclonal antibodies (Immunotech, Westbrook, Maine) by a standard dual-color flow cytometry technique [19].

Statistical Analysis

Continuous variables were compared by using the Wilcoxon matched-pairs signed-rank test. Trends for possible dose-response relation were evaluated using the Spearman rank correlation coefficients (r_S) between dose and outcome.

Industry Role

Thrombopoietin and partial funding for the study were provided by Genentech, Inc. The study was a collaborative effort between the principal investigator and the industrial sponsor. Data collection, data analysis, the writing of the manuscript, and the decision to publish the manuscript were under the control of the principal investigator. The manuscript was reviewed by the industrial sponsor before submission.

Results

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Twelve chemotherapy-naive patients (7 men and 5 women) with sarcoma of diverse histologic sub-types were entered into the dose-ranging portion of this phase I trial, which studied thrombopoietin before chemotherapy. All patients were considered evaluable for clinical tolerance and response to thrombopoietin. The median age of these patients was 42 years (range, 16 to 63 years), and the median Karnofsky performance status score was 90 (range, 80 to 100). Four patients had previously received radiation therapy, and eight had previously had surgery.

Peripheral Blood Counts

Treatment with a single dose of thrombopoietin was associated with increases (1.3-fold to 3.6-fold) in platelet counts (baseline mean, 264 x 10⁹/L; maximal mean, 592 x 10⁹/L) (P = 0.002). The increase in platelet count was seen at all dose levels (Figure 1) in all patients. The peak response in platelet count was dose-related (Figure 1); increases from baseline of 61%, 107%, 118%, and 212% were seen at the 0.3, 0.6, 1.2, and 2.4 µg/kg dose levels, respectively (r_S = 0.720, P = 0.01). At the highest dose level tested (2.4 µg/kg), the platelet count increased to approximately 1 million or more in two of three patients treated (970 x 10⁹/L in one patient and 1392 x 10⁹/L in the other).

View larger version (15K): [in this window] [in a new window]   Figure 1. Dose effect of thrombopoietin (TPO) on circulating platelet counts (left) and bone marrow megakaryocytes (right). Percentage increases from baseline after various doses of thrombopoietin are shown. Data given are the mean + SE for all patients at each dose level.

An increase in platelet count was seen early (Figure 2); 10 of 12 patients showed increases from baseline by day 4 (mean increase, 1.2-fold; P = 0.005), and all patients showed increases from baseline by day 8 (mean increase, 1.8-fold; P = 0.002). Platelet counts peaked on median day 12 (range, 10 to 15 days) (mean increase, 2.2-fold; P = 0.002). After maximum response, the platelet counts gradually declined toward baseline. By day 21 (before the initiation of chemotherapy), the platelet counts were still higher than baseline, especially at the highest dose level (at which they were increased 1.5-fold).

View larger version (23K): [in this window] [in a new window]   Figure 2. Kinetics of platelet response after a single dose (arrows) of thrombopoietin. The data from each patient treated at four different dose levels are shown.

Overall platelet morphology appeared normal after treatment. At the time of peak platelet counts, the mean platelet volume was slightly lower than baseline (7.6 fL compared with 8.5 fL); by day 21, it was close to baseline (8.2 fL).

Platelet Functions

To determine whether platelets derived after thrombopoietin treatment were capable of mediating functions critical for hemostasis, studies of platelet aggregation in response to three agonists were done on platelets obtained from patients both before and after thrombopoietin treatment (day 7) and from normal controls (Table 1). No statistically significant changes in platelet aggregation were seen in relation to baseline or control samples after thrombopoietin treatment in response to adenosine diphosphate, collagen, or thrombin (P > 0.2).

Other Hematopoietic Cell Lineages

Although a slight upward trend was seen in leukocyte counts after thrombopoietin treatment, the counts remained within normal limits; these changes were not clinically significant. Similarly, hemoglobin values did not change at any dose level. A minor overall decrease in hemoglobin level was seen with time (baseline mean, 13.4 ± 0.4; mean at day 21, 12.8 ± 0.4) that was probably related to the phlebotomies that were done frequently during the study.

Pharmacokinetics

Before treatment, endogenous serum thrombopoietin was detectable (0.096 to 0.24 ng/mL) in 4 of 12 patients. After thrombopoietin administration, the initial maximum thrombopoietin concentrations were proportional to the dose and ranged from 5 to 50 ng/mL (Figure 3). Thrombopoietin concentrations declined more slowly after the higher doses. For example, at 6 hours, the mean serum thrombopoietin levels had declined by 50% after the 2.4-µg/kg dose and by 80% after the 0.3-µg/kg dose. The terminal serum half-life for the higher doses (1.2 to 2.4 µg/kg) ranged from 18 to 32 hours. Overall, thrombopoietin remained in the circulation for 5 to 6 days after intravenous bolus administration.

View larger version (20K): [in this window] [in a new window]   Figure 3. Serum thrombopoietin (TPO) concentrations after intravenous bolus administration of thrombopoietin at different dose levels. Data given are the mean + SE for three patients per dose level.

Clinical Tolerance

Treatment with thrombopoietin was well tolerated by all patients, and no significant adverse events were attributed to thrombopoietin. Six patients reported occasional mild headaches in their symptom-record diaries, but these headaches were not closely associated with the timing of thrombopoietin administration. No local skin reactions, bone pain, constitutional symptoms, weight gain, edema, or changes in chemistry related to the study drug were seen during this period.

Antibody Analysis

To determine whether thrombopoietin therapy was associated with the development of antibodies that might interfere with the hematopoietic effect of thrombopoietin, serum samples from all patients were screened weekly. Serum samples obtained on days 13 and 22 after dosing from 1 of the 12 patients tested positive for antibodies reactive to full-length thrombopoietin. These antibody titers were transient, were non-neutralizing, and were not associated with clinical sequelae.

Effects on Bone Marrow

To better understand the cellular basis for the hematopoietic response to thrombopoietin, we examined the bone marrow both before and 7 days after thrombopoietin treatment for expansion of progenitor cells, lineage specificity, and maturation.

Bone Marrow Morphology

The increase in platelet count was associated with a significant increase in the number of bone marrow megakaryocytes (P = 0.003) in a dose-related manner (Figure 1); it ranged from a 1.2-fold increase at the lowest dose level to a 4-fold increase at the highest dose level (r_S = 0.867, P < 0.001). Although most megakaryocytes showed normal morphology, several large megakaryocytes with abundant cytoplasm and multilobulated nuclei were identified (Figure 4). Overall, the size of the megakaryocytes increased significantly from baseline (from a mean diameter of 36 µm [range, 17 to 64 micro m] to a mean of 44 µm [range, 20 to 87 micro m]; P < 0.001).

View larger version (124K): [in this window] [in a new window]   Figure 4. Top. Photoµgraphs of a section of bone marrow from a patient before (left) and after (right) thrombopoietin treatment. Megakaryocytes are essentially normal in morphologic appearance; some are increased in size. Abundant cytoplasm and multilobulated nuclei are apparent. Bottom. Masson trichrome-stained section of a bone marrow biopsy specimen. A markedly increased number of megakaryocytes is apparent, but collagen content after thrombopoietin treatment (right) is not increased compared with baseline (left).

The granulocytic and erythroid series showed normal maturation without significant changes in the myeloid:erythroid cell ratio or bone marrow cellularity (Table 2). No significant increase in bone marrow collagen (Figure 4, bottom) was identified after thrombopoietin treatment in any of the specimens examined.

Megakaryocyte Ploidy Analysis

To determine whether thrombopoietin treatment affected megakaryocyte maturation, bone marrow megakaryocyte ploidy was assessed at all dose levels. Although the modal ploidy peak remained at 16N, increased percentages of megakaryocytes were seen at lower ploidy classes (P < 0.001 for 4N and 8N) at all dose levels (Figure 5). In addition, a trend toward increased higher ploidy levels was seen at the higher doses (P = 0.06 for 64N and P = 0.06 for 128N). Thus, thrombopoietin treatment enhanced both maturation of megakaryocytes and expansion of more immature megakaryocytes.

View larger version (29K): [in this window] [in a new window]   Figure 5. Bone marrow megakaryocyte DNA ploidy by flow cytometry before (white bars) and 7 days after (striped bars) thrombopoietin treatment. Top. Data given are the mean + SE for patients treated at all dose levels (n = 9). Bottom. Data given are the mean + SE for four of five evaluable patients treated at the two highest dose levels. The fifth patient had a higher ploidy distribution at the start of treatment and is not included here.

Bone Marrow Progenitors and CD34⁺ Cells

To determine whether thrombopoietin had a lineage-specific effect at the progenitor cell level, bone marrow progenitor cell frequencies were assayed for megakaryocyte (CFU-MK and BFU-MK), myeloid (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) cells. Frequency of bone marrow CFU-MK (2.7-fold to 33-fold) was increased in 5 of 11 patients examined. A significant increase was also seen in the number of mature subsets of myeloid (1.8-fold; P = 0.005) and erythroid (1.7-fold; P = 0.016) progenitors, and similar trends were seen for the immature subsets of myeloid and erythroid progenitors (data not shown) and multipotential progenitors (Figure 6). The percentage of bone marrow cells in S-phase was also significantly increased (P = 0.013) from baseline (Figure 6). In addition, the percentage of bone marrow CD34⁺ (P = 0.012) and CD41⁺ (P = 0.002) cells increased an average of 2-fold in number (Table 2).

View larger version (33K): [in this window] [in a new window]   Figure 6. Frequency (left) and cycling status (right) of bone marrow myeloid (colony-forming unit-granulocyte-macrophage [CFU-GM]), erythroid (burst-forming unit-erythroid [BFU-E]), and multipotential (colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte [CFU-GEMM]) progenitors before and after thrombopoietin treatment. Data given are the mean ± SE for all patients studied.

Effects on Mobilization of Progenitors

To determine whether the increase in bone marrow progenitors was associated with a mobilization effect, the peripheral blood was assayed for progenitor cells. A significant increase was seen in the number of progenitors of myeloid (CFU-GM), erythroid (BFU-E), and myelo-erythroid (CFU-MIX) cells. Although individual patient responses varied, a dose-dependent trend was seen (Figure 7). At the highest dose level, a mean maximum increase of progenitor cell frequencies was seen: as high as 5.7-fold for CFU-GM, 7.8-fold for BFU-E, and 10-fold for CFU-MIX.

View larger version (18K): [in this window] [in a new window]   Figure 7. Effect of thrombopoietin treatment on mobilization of myeloid (colony-forming unit-granulocyte-macrophage [CFU-GM]), erythroid (burst-forming unit-erythroid [BFU-E]), and myelo-erythroid (CFU-MIX) progenitors at 0.3 (open bars), 0.6 (striped bars slanting up), 1.2 (black bars), and 2.4 (striped bars slanting down) µg/kg of body weight. Data shown represent the peak response (mean + SE), which was seen between days 3 and 7.

Discussion

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Over the past decade, the recognition that the myeloid growth factors control the production of neutrophils (G-CSF and GM-CSF) and erythrocytes (erythropoietin) has led to the clinical application of these factors for the management of patients with cancer who are undergoing myelosuppressive therapy [20-23]. Thrombocytopenia has been managed primarily by platelet transfusions and chemotherapy dose modifications and has prompted a vigorous search for an agent that regulates platelet production. Several thrombopoietic cytokines have shown modest activity, but most of these mediate pleiotropic biological effects, and their use is frequently associated with significant side effects [24-29].

After an almost 40-year search for a bona fide humoral regulator of platelet production, thrombopoietin has been identified, cloned, and made available for clinical testing [4-9, 30]. Our objective was to evaluate the in vivo biological effects of thrombopoietin, including clinical tolerance and pharmacodynamics, and to define the nature of the hematopoietic response to this cytokine in patients with cancer who had normal hematopoietic function before receiving chemotherapy. We show that treatment with a single dose of thrombopoietin produced a marked increase in circulating platelet counts in a dose-related manner. Platelet response (increases of 1.3-fold to 3.6-fold) was seen in all patients, including the cohort that received the lowest dose of thrombopoietin. The platelets produced in response to thrombopoietin had normal morphologic appearance and displayed normal aggregation function in response to various stimuli. Thus, our findings indicate that thrombopoietin is a potent stimulus for the production of platelets with normal function in humans.

We observed several interesting facets of the hematopoietic response to thrombopoietin in these patients. The increase in platelet count was seen by day 4 in most patients, suggesting that the initial component of the response was related to enhanced maturation of megakaryocytes, platelet release, or both. Platelet counts continued to increase, however, reaching a peak between days 10 and 15. This suggests that a second component of the response might be related to an expansion of the megakaryocyte compartment. After the maximum response, platelet counts declined toward baseline. However, platelet counts at day 21 before the initiation of chemotherapy were still higher than baseline counts, especially at the highest dose level; this finding indicates a prolonged biological effect.

This sustained response after a single dose of thrombopoietin can be explained by several possible mechanisms. First, we observed that thrombopoietin has a prolonged serum half-life of about 20 to 30 hours and remains in the circulation for about 5 to 6 days. Furthermore, although peak serum concentration was proportional to dose, the terminal half life was prolonged at higher doses, perhaps because of the saturation of the thrombopoietin clearance process (thought to occur primarily in platelets by receptor-mediated endocytosis) [31].

A second explanation might be related to the proliferative effect of thrombopoietin at the bone marrow level. Some patients had an increase in the number of megakaryocytic progenitors (CFU-MK). The proliferative effect of thrombopoietin on bone marrow was also marked by a twofold increase in the percentage of CD34⁺ cells and a concomitant twofold increase in CD41⁺ subsets, reflecting a selective maturation along the megakaryocytic pathway. In fact, the number of megakaryocytes increased in proportion to the dose, and the megakaryocytes displayed normal morphology. Moreover, ploidy analysis indicated not only the maturation effect of thrombopoietin but also the expansion of immature subsets of megakaryocytes, which may be related to the proliferative effect. Third, because the life span of platelets is 9 to 10 days, the response in number of platelets would be expected to last for several days.

The proliferative effect of thrombopoietin on bone marrow was not limited to the megakaryocytic lineage. A significant increase was seen in the frequency of progenitors of myeloid, erythroid, and multipotential cell lineages. Furthermore, the proportion of progenitors in S-phase also increased after treatment with thrombopoietin. Despite this multilineage stimulatory effect of thrombopoietin on bone marrow progenitors, no major effect was seen on peripheral leukocyte counts or hematocrit values. Thus, the proliferative effect at the progenitor level appeared to be coupled with differentiation primarily along the megakaryocytic pathway.

Despite the expansion of the progenitor pool in bone marrow, the overall cellularity remained unchanged, possibly because of the extramedullary release of progenitors into the peripheral blood. One of the biological properties of hematopoietic growth factors that has demonstrable clinical effect is the ability to mobilize hematopoietic progenitors that can be harvested and reinfused after dose-intensive or myeloablative therapy [32-34]. In our study, a single dose of thrombopoietin strongly mobilized the progenitors of multiple lineages. This apparent increase in the circulating pool of progenitor cells was further supported by a several-fold increase in very primitive hematopoietic cells (CD34⁺/Thy-1⁺/Lin^-) in the peripheral blood [35].

Treatment with thrombopoietin was well tolerated by all patients; no significant adverse events related to thrombopoietin or thrombocytosis were seen. Unlike other cytokines, thrombopoietin was not associated with fever, constitutional symptoms, bone pain, inflammatory symptoms, fluid retention, or any organ toxicity [24-29]. Thus, the lack of significant toxicity and the lineage-dominant biological effect of thrombopoietin may provide a better therapeutic ratio than do other cytokines with thrombopoietic potential.

One potential concern associated with inducing thrombocytosis is an increased risk for thrombosis, which is frequently seen in patients with myeloproliferative disorders. However, the lack of morphologic and functional platelet abnormalities and the transient nature of induced thrombocytosis may limit risk in this clinical setting.

In summary, our findings indicate that thrombopoietin is a potent stimulator of megakaryocytopoiesis and thrombopoiesis in humans. Thrombopoietin administered as a single dose produced prolonged increases in circulating platelet counts. Although it had a lineage-dominant effect on peripheral blood counts, it mediated a multilineage effect at the bone marrow progenitor cell level and mobilized progenitors of multiple lineages into the peripheral blood.

These observations have several potential ramifications. For example, in a manner analogous to that of the clinical application of myeloid growth factors, thrombopoietin has the potential to attenuate chemotherapy-induced thrombocytopenia. Early results of the trials using this form of thrombopoietin [36] as well as a polyethylene glycol-conjugated form of the molecule [37, 38] demonstrate such clinical activity. However, previous studies with growth factors indicate the critical importance of the timing of growth factor administration in relation to chemotherapy to achieve maximal clinical benefit [39-41]. In this regard, the studies reported here show that the peak platelet response occurs between day 10 and day 15. Thus, treatment with thrombopoietin before chemotherapy may provide a holdover period until platelet production is 1) resumed after chemotherapy and 2) possibly further enhanced by thrombopoietin given after chemotherapy. Studies are being done to determine the optimal schedule of thrombopoietin administration.

A second intriguing possibility is that the potent biological effect of a single dose will allow thrombopoietin to be given to patients or normal donors not only to mobilize progenitor cells for peripheral stem cell transplantation but also to collect platelets that can be transfused as an apheresis product, thus potentially reducing the risk for alloimmunization associated with exposure to multiple donors. It should be noted, however, that our trial involved a small, defined group of patients with sarcoma who had unperturbed normal hematopoiesis. Future trials should investigate the potential roles of this agent in the prevention and treatment of thrombocytopenia associated with intrinsic or compromised hematopoietic disorders.

Dr. Murray: SyStemix, 3155 Porter Drive, Palo Alto, CA 94304.

Dr. Bueso-Ramos: Hematology, Box 72, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.

Drs. Patel, Papadopolous, and Benjamin: Melanoma/Sarcoma, Box 077, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.

Dr. Hoots: Pediatrics, Box 087, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.

Dr. Hittelman: Clinical Investigation, Box 019, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.

Dr. Johnston: Biomathematics, Box 237, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.

Mr. Yang and Drs. Paton, Cohen, and Hellmann: Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080-4990.

Dr. Broxmeyer: Indiana University School of Medicine, Walther Oncology Center, Medical Research and Library Building, 975 West Walnut Street, 5th Floor, Indianapolis, IN 46223-5121.