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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2798-2806
Recombinant Human Thrombopoietin in Combination With Granulocyte
Colony-Stimulating Factor Enhances Mobilization of Peripheral Blood
Progenitor Cells, Increases Peripheral Blood Platelet Concentration,
and Accelerates Hematopoietic Recovery Following High-Dose Chemotherapy
By
George Somlo,
Irena Sniecinski,
Anna ter Veer,
Jeffrey Longmate,
Gaylord Knutson,
Stanimir Vuk-Pavlovic,
Ravi Bhatia,
Warren Chow,
Lucille Leong,
Robert Morgan,
Kim Margolin,
James Raschko,
Stephen Shibata,
Merry Tetef,
Yun Yen,
Stephen Forman,
Dennie Jones,
Mark Ashby,
Gwen Fyfe,
Susan Hellmann, and
James H. Doroshow
From the Departments of Medical Oncology and Therapeutics
Research, Transfusion Medicine, and Biostatistics, City of Hope
National Medical Center, Duarte, CA; Stem Cell Laboratory, Mayo Clinic
Cancer Center, Rochester, MN; and Genentech Inc, South San Francisco,
CA.
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ABSTRACT |
Lineage-specific growth factors mobilize peripheral blood progenitor
cells (PBPC) and accelerate hematopoietic recovery after high-dose
chemotherapy. Recombinant human thrombopoietin (rhTPO) may further
increase the progenitor-cell content and regenerating potential of PBPC
products. We evaluated the safety and activity of rhTPO as a PBPC
mobilizer in combination with granulocyte colony-stimulating factor
(G-CSF) in 29 breast cancer patients treated with high-dose chemotherapy followed by PBPC reinfusion. Initially, patients received
escalating single doses of rhTPO intravenously (IV) at 0.6, 1.2, or 2.4 µg/kg, on day 1. Subsequent patients received rhTPO 0.6 or 0.3 µg/kg on days  3, 1, and 1, or 0.6 µg/kg on days 1 and 1. G-CSF, 5 µg/kg IV or subcutaneously (SC) twice daily, was started on
day 3 and continued through aphereses. Twenty comparable, concurrently
and identically treated patients (who were eligible and would have been
treated on protocol but for the lack of study opening) mobilized with
G-CSF alone served as comparisons. CD34+ cell yields were
substantially higher with the first apheresis following rhTPO and G-CSF
versus G-CSF alone: 4.1 × 106/kg (range, 1.3 to 17.6)
versus 0.8 × 106/ kg (range, 0.3 to 4.2), P = .0003. The targeted minimum yield of 3 × 106
CD34+ cells/kg was procured following a single apheresis
procedure in 61% of the rhTPO and G-CSF-mobilized group versus 10%
of G-CSF-mobilized patients (P = .001). In rhTPO
and G-CSF mobilized patients, granulocyte (day 8 v 9, P
= .0001) and platelet recovery (day 9 v 10, P
= .07) were accelerated, and fewer erythrocyte (3 v 4, P = .02) and platelet (4 v 5, P = .02)
transfusions were needed compared with G-CSF-mobilized patients.
Peripheral blood platelet counts, following rhTPO and G-CSF, were
increased by greater than 100% and the platelet content of PBPC
products by 60% to 110% on the first and second days of aphereses
(P < .0001) with the greatest effect seen with repeated
dosing of rhTPO at 0.6 µg/kg. rhTPO is safe and well tolerated as a
mobilizing agent before PBPC collection. Mobilization with rhTPO and
G-CSF, in comparison to a comparable, nonrandomized G-CSF-mobilized
group of patients, decreases the number of apheresis procedures
required, may accelerate hematopoietic recovery, and may reduce the
number of transfusions required following high-dose chemotherapy for
breast cancer.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THROMBOPOIETIN, the primary regulator of
megakaryocyte development and the ligand for the cytokine receptor
C-Mpl, has been cloned by several groups of
investigators.1-5 C-Mpl is expressed on the surface of
cells of megakaryocytic lineage and other hematopoietic progenitors;
once activated, it can promote proliferation and maturation of
megakaryocytes,6 and progenitors committed to the
myelomonocytic7 and erythroid lineage.8
High-dose chemotherapy and stem-cell rescue has been increasingly used
for the treatment of a variety of solid tumors and hematologic
malignancies.9,10 Administration of colony-stimulating factors such as granulocyte colony-stimulating factor (G-CSF) and
granulocyte-macrophage colony-stimulating factor (GM-CSF) in the
setting of peripheral blood progenitor cell (PBPC) mobilization and
apheresis has shortened the duration of potentially life-threatening pancytopenia following high-dose chemotherapy.11,12
However, some patients will mobilize PBPC poorly with G-CSF or GM-CSF. In addition, a fraction of patients will become refractory to platelet
transfusions despite the fact that the use of filtered platelets and
changes in the quantitative indication for transfusion have diminished
exposure to potentially immunogenic antigens.13,14
In the preclinical setting, a single injection of recombinant human
thrombopoietin (rhTPO) into Rhesus monkeys decreased radiation-induced thrombocytopenia, while coadministration of rhTPO and G-CSF or GM-CSF
resulted in accelerated neutrophil recovery.15
Adenovirus-mediated transfer of the TPO gene given intraperitoneally
into mice before exposure to radiation and carboplatin ameliorated
thrombocytopenia.16 In the clinical setting, a single dose
of rhTPO before chemotherapy resulted in expansion of all progenitor
lines and a dose-related increase in the megakaryocyte pool; peripheral
blood platelet counts increased 4 days after rhTPO injection with a
peak between days 10 and 15.17
Based on favorable preclinical and clinical safety data, we set out to
test the safety and efficacy of escalating single and multiple doses of
rhTPO as a mobilizing agent before the procurement of PBPC.
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MATERIALS AND METHODS |
Patients.
All patients gave their written, voluntary informed consent. This study
was approved by the Institutional Review Board of the City of Hope
National Medical Center (Duarte, CA).
Between July 1996 and November 1997, 29 patients with responding stage
IV or high-risk stage II/III primary breast carcinoma were accrued.
Patients with a Karnofsky performance status greater than 80%,
adequate organ function, left ventricular cardiac ejection fraction
 55%, and a measured creatinine clearance 80 mL/min were eligible.
The use of aspirin, nonsteroidal antiinflammatory agents, and
anticoagulant drugs was not allowed (except to maintain line patency).
Chemotherapy or radiation therapy within 3 weeks before mobilization,
or the use of growth factors other than G-CSF, was prohibited.
Study design.
Patients had a double-lumen Hickman catheter (Bard Access
Systems, Salt Lake City, UT) inserted before mobilization of PBPC. Safety of rhTPO, at any given dose level, had to be documented in three
patients before accrual began at the next dose level. Initially, groups
of patients received a single intravenous (IV) injection of rhTPO on
day 1, escalating the dose through 0.6, 1.2, or 2.4 µg/kg (arm A).
Completion of accrual on arm A was required before accrual to the
subsequent cohort. Subsequent patients received rhTPO on days 3,
1, and 1 at doses of 0.6 µg/kg or 0.3 µg/kg (arm B) or rhTPO
0.6 µg/kg on days 1 and 1 (arm C). G-CSF (Neupogen; Amgen,
Thousand Oaks, CA) 5 µg/kg twice daily, subcutaneously (SC) or IV was
started on day 3 and continued through aphereses. rhTPO (Genentech Inc,
South San Francisco, CA), a full-length glycosylated molecule, was
reconstituted in preservative-free normal saline before injection.
Aphereses were started on the fourth day of G-CSF administration (day
6) using a Spectra (Cobe Laboratories, Lakewood, CO), or Fenwall
CS-3000 Plus cell separator (Baxter, Deerfield, IL); approximately 10 L
of blood was processed during each collection. The mobilization schema
is illustrated in Fig 1. A minimum target of 3 × 106 CD34+ cells/kg (and 7 × 108 mononuclear cells/kg) was procured. A cohort of
20 concurrently treated patients were mobilized with G-CSF 5 µg/kg
twice per day, SC or IV, daily. These patients were eligible and
otherwise similar to those on-study and would have been treated on
protocol but for the lack of study openings at the time of their
evaluation. Starting on the fourth day of G-CSF administration, the
duration and procedure of apheresis were identical to those of the
rhTPO study group; 7 × 108 mononuclear cells/kg
were collected. PBPC were cryopreserved by simple immersion in a
130°C freezer.18
High-dose chemotherapy.
Patients received both cisplatin 125 mg/m2 and etoposide 30 mg/kg on days 12 and 5 followed by cyclophosphamide 100 mg/kg on day 3. PBPC were reinfused on a divided schedule: 25%
of PBPC were infused on day 2 following high-dose chemotherapy
and 75% were infused on day 0. All patients received G-CSF 5 µg/kg
twice daily IV from day 2 through absolute granulocyte recovery
to 1,500/µL.18 Blood products were filtered and
irradiated at 2,500 cGy and were transfused to maintain a platelet
count of greater than 20 × 109/L and a hematocrit of
25 or as clinically indicated.19
Patient monitoring.
Patients were monitored for adverse reactions with each injection of
rhTPO. Total WBC, differential, and platelet counts were measured on
the days of rhTPO and G-CSF administration and during aphereses. Serum
chemistries, coagulation parameters, and samples for anti-rhTPO
antibodies were monitored during the mobilization, and following
high-dose chemotherapy and PBPC reinfusion (transplant) phase. No more
than two episodes of grade 3 toxicity were allowed at any dose level;
any grade 4 toxicity related to the administration of rhTPO defined the
maximum-tolerated dose of rhTPO. Development of neutralizing antibodies
to rhTPO or a platelet count greater than 600,000/µL persisting
beyond 24 hours were also considered dose-limiting toxicities.
Hematologic end points.
The number of days needed to reach an absolute granulocyte count 0.5 × 109/L and platelet independence (defined as the day
after the last platelet transfusion) were calculated from day 0 of the
"transplant phase." The number of erythrocyte and platelet
transfusions required were tabulated. Single apheresis and pooled
platelet products were each counted as one product.
Flow cytometry studies.
PBPC apheresis or peripheral blood samples were stained with either
phycoerythrin (PE)-conjugated anti-CD34 monoclonal antibody and dual
stained with anti-CD45-fluorescein isothiocyanate (FITC) or, for
assessment of the presence of megakaryocytic lineage samples, were
stained with anti-CD34-PE/anti-CD41-FITC (Becton Dickinson, San Jose,
CA). Samples of 300 µL containing approximately 1 million cells were
incubated with antibody for 30 minutes at 4°C. After the addition
of 2 mL erythrocyte lysing solution (Ortho-mune; Ortho Diagnostic
Systems, Raritan, NJ), samples were incubated for 10 minutes at room
temperature. Cells were then washed twice in phosphate-buffered saline
(PBS) Buffer Reagent (Coulter, Miami, FL) and resuspended in 500 µL
PBS. Analysis was performed using a FACSCalibur or FACSort flow
cytometer equipped with argon-ion laser, tuned at 488 nm (Becton
Dickinson), and analyzed with Cellquest 3.1 software (Becton
Dickinson); 100,000 cells were acquired for each analysis. CD34/CD45
analysis was performed as described earlier.18 For the
CD34/CD41 analysis, the primary gate was set up using forward scatter
versus side scatter. All CD34+ cells were analyzed for
side-scatter properties and expression of CD41; subsequently,
CD34+ cells within the low side-scatter population were
further analyzed for CD41 expression.
Progenitor-cell assays.
Peripheral blood was collected in heparin before each injection of
rhTPO and on the days of apheresis. PBPC were diluted 1:5 with
Iscove's modified Dulbecco's medium (IMDM) in heparin. Samples were
shipped at room temperature overnight to the Stem Cell Laboratory (Mayo
Clinic Cancer Center, Rochester, MN).
Mononuclear cells were isolated by density gradient centrifugation in
Lymphocyte Separation Medium (LSM; Cappel, ICN Biomedical, Aurora, OH; 1.077 g/mL) and diluted with IMDM to no more than 5 × 106 cells/mL. Eight milliliters of cell suspension was
centrifuged at 400g for 30 minutes at 22°C. Cells at the
interface were collected and washed twice with 3 vol of IMDM.
Colony-forming unit-granulocyte, macrophage (CFU-GM), colony-forming
unit-granulocyte, erythrocyte, megakaryocyte, macrophage (CFU-GEMM) and burst-forming unit-erythroid (BFU-E) were
assayed in a methylcellulose medium (MethoCult GF H4434; StemCell
Technologies, Vancouver, Canada). Cells were plated at a final cell
density of 1 or 2 × 105/mL (peripheral blood) and 1 or 3 × 104/mL (PBPC). After 14 days at 37°C in
5% CO2, the colonies were identified by morphology and
scored. Colony-forming unit-megakaryocyte (CFU-MK) was assayed in an
agarose medium (MegaCult; StemCell Technologies); cells were plated at
a final density of 4 × 105/mL (peripheral blood) or 1 × 105/mL (PBPC). After 18 days at 37°C in 5%
CO2, the colonies were fixed with methanol-acetone and
immunohistochemically detected by the anti-GPIIb/IIIa antibody and
alkaline phosphatase detection system supplied by the manufacturer.
Statistical analysis.
The study was primarily designed to identify tolerable doses and
schedules of rhTPO. Comparison of rhTPO doses and schedules, and
comparison to patients not receiving rhTPO was planned to evaluate
hematologic response in the context of a phase I study, and not to
achieve any specified power. The G-CSF-only mobilized group consisted
of comparable, concurrently treated patients who were eligible to
enroll on study but did not for lack of an opening on the trial.
Nonevaluable patients were excluded from analysis as described in the
Results. Patients receiving rhTPO were combined in a single group for
comparison to G-CSF-only patients using rank-sum tests; an F test was
applied for overall comparison between these two groups after
stratifying for prior exposure to radiation therapy. Comparisons among
rhTPO regimens were evaluated by Kruskal-Wallis tests20;
95% confidence intervals (CIs) for percent (fold) differences were
based on t statistics for log-percent difference.21
The effects of dose and schedule were evaluated by fitting multiple regression models, using logarithms of responses as described in the
Results. Separate regressions were fit to the first and second days of
cell collections to evaluate the relationship between platelet content
for stem cell products and peripheral blood platelet counts.
Correlations and coefficients of determination are provided as rough
summaries. An F test was applied to verify the presence of significant correlation.
| |
RESULTS |
The effects of rhTPO and G-CSF on PBPC mobilization were assessable in
26 of 29 patients (two patients were removed from the study due to
bacteremia associated with Hickman catheter infection; one patient who
had experienced prolonged leukocytopenia after prior standard
chemotherapy mobilized so poorly that she was taken off study).
Twenty-three of the 26 patients were assessable both during the
mobilization and the "transplant" phase; three patients were not
evaluable during the transplant phase due to delay of their high-dose
chemotherapy (progression, n = 1; superior vena cava occlusion [likely
due to a previous catheter infection], n = 1; bacteremia after
apheresis, n = 1).
Characteristics of the 26 assessable rhTPO- and G-CSF-treated patients
and 20 concurrently and identically treated G-CSF-only mobilized
patients are listed in Table 1. Most
characteristics, such as age, stage, prior chemotherapeutic exposure,
and number of days between apheresis and PBPC reinfusion were similar
except for a higher percentage of G-CSF-only-mobilized group (control group) having been exposed to prior radiation treatment.
Table 2 demonstrates the CD34+
cell (CD34+/CD45+) yield procured during the
first apheresis, the number of aphereses needed to obtain 3 × 106 CD34+ cells/kg, and transfusion
requirements in the combined cohorts of patients mobilized with rhTPO
and G-CSF versus the control group mobilized with G-CSF alone and
stratified by prior exposure to radiation therapy. The number of
apheresis procedures needed to procure 3 × 106/kg
CD34+ cells was lower in the group mobilized with rhTPO and
G-CSF (2 [range, 1 to 3] v 3 [range, 1 to 5];
P < .0001). The cumulative CD34+ cell
yield never reached 3 × 106 /kg in 7 of 20 patients mobilized with G-CSF alone, although the required
mononuclear-cell target for patients in the control group was reached
by all. The number of actual aphereses performed (targeted originally
to procure 3 × 106/kg CD34+ cells in
the rhTPO/G-CSF group and >7 × 108 mononuclear
cells/kg in the G-CSF-mobilized group) was fewer in the
rhTPO-mobilized group (median, 2 [range, 2 to 3] v 3 [range, 2 to 6]; P < .0001, data not shown).
In 16 of 26 patients in the rhTPO-mobilized group, the targeted yield
of 3 × 106 CD34+ cells/kg was reached
with the first apheresis procedure versus in only 2 of 20 G-CSF
mobilized patients ( 2, P < .001), apheresis
from three additional patients in the G-CSF-only group resulted in 3 × 106 CD34+ cell yield/kg on days 5 (n = 2) and 6 (n = 1), while no patient in the control group yielded 3 × 106 CD34+ cells/kg beyond day 6 (data
not shown). In the G-CSF-mobilized group, the highest numbers of
CD34+ cells were procured during the first apheresis (day 4 of G-CSF) in 8 and during the second procedure (day 5 of G-CSF) in 5 patients; CD34+ cell yield from 7 patients peaked evenly
between days 6 and 8. The median CD34+ yield was 4.1 × 106/kg (range, 1.3 to 17.6 × 106/kg) in products collected on the first scheduled day of
apheresis from patients in the rhTPO-mobilized group versus 0.8 × 106/kg (range, 0.3 to 4.2 × 106/kg) in
the concurrently treated and G-CSF-mobilized control group. A 3.5-fold
increase in CD34+ cell content per apheresis (95% CI,
2.7-to 6.6-fold increase in geometric mean) following administration of
rhTPO was observed: the median number of (range) CD34+
cells per kilogram per apheresis was 3.8 × 106 (1.1 to 12.7) in the rhTPO-mobilized group versus 1.1 × 106 (0.5 to 4.7) in the control group (P = .014).
Higher yields of mononuclear cells were procured following
administration of rhTPO: 3.7 × 108 mononuclear
cells/kg/apheresis (range, 0.5 to 6.5) versus 2.8 × 108 mononuclear cells/kg/apheresis (range, 1.6 to 4.5). We
observed an approximate 30% increase in the mononuclear-cell yield per apheresis (95% CI, 16% to 59% increase in geometric mean), with a
statistically significant difference seen even after stratification for
the possible effects of prior radiation treatment (P = .04 data
not shown). Both RBC (3 v 4, P = .02) and platelet (4 v 5, P = .02) transfusion requirements were decreased
in the rhTPO-mobilized group compared with G-CSF-mobilized controls.
Hematopoietic recovery was also faster in the rhTPO group: granulocyte
and platelet recoveries were accelerated, as illustrated in Fig
2A and B (8 v 9 days [P = .0001] and 9 v 10 days [P = .07], respectively).
Progenitor-cell yield and hematopoietic recovery were improved
following mobilization with the rhTPO/G-CSF combination versus G-CSF
alone, in patients with or without prior exposure to radiation therapy.
Figure 3 displays the mean
CD34+ yield for cohorts of patients receiving different
doses and/or schedules of rhTPO on arms A, B, and C. The Kruskal-Wallis
test suggested a dose/schedule dependent effect (P = .03); the
highest yield (15.1 × 106/kg v 7.5 × 106/kg) was observed when three doses of rhTPO 0.6 µg/kg
were administered every other day (P = .004, F test).
Table 3 compares the effects of rhTPO/G-CSF
mobilization versus G-CSF alone on peripheral blood platelet counts on
the days of aphereses and depicts the platelet content of each
apheresis product. We observed an approximately 100% and 130%
increase in peripheral blood platelet counts on the first and second
days of apheresis and 60% and 110% increase in platelet yields in the apheresis products after mobilization with rhTPO and G-CSF, in comparison to mobilization with G-CSF only.
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Table 3.
Platelet Content of PBPC Products and Peripheral Blood
Platelet Concentration on the First and Second Days of Apheresis by
Mobilizing Regimen
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Table 4 depicts the mean platelet
concentrations in peripheral blood on the first 2 days of apheresis (5 and 6 days after the last dose of rhTPO) and illustrates the total
platelet content of the combined apheresis products sorted by the
different doses/ schedules of rhTPO. Both peripheral blood platelet
counts (P = .01) and total platelet content
(P = .02) were highest in patients mobilized with three doses
of rhTPO at 0.6 µg/kg, consistent with schedule- and dose-dependent
effects. Platelet content of apheresis products was directly related to
peripheral blood platelet concentration, as shown in Fig
4.
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| Fig 4.
Total platelet content of PBPC products in the PBPC
products collected on the first and second days of apheresis in
relationship to peripheral blood platelet concentration.
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Table 5 describes the percentage of CD41+
cells within the CD34+ cell population: increased
percentages of CD41+ cells were seen following
administration of either single or repeated injections at the higher
(1.2 to 2.4 µg/kg) rhTPO doses. We observed the highest percentage of
CD41+ cells within the CD34+ population
following sequential administration of rhTPO at 0.6 µg/kg (P = not significant [NS]).
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Table 5.
Percentage of CD41+ Cells Within the
CD34+ Low Side-Scatter Population From PBPC Products by
rhTPO Dose Level
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We analyzed the effect of different rhTPO doses and schedules on
CFU-GM, CFU-GEMM, CFU-MK, and BFU-E formation from peripheral blood
samples. We found a significant increase over baseline in the number of
CFU-GM (5.15-fold mean increase; range, 0.9 to 31; 95% CI, 3.6 to
7.5), CFU-GEMM (2.5-fold mean increase; range, 0.4 to 15; 95% CI, 1.7 to 3.6), and CFU-MK (2.5-fold increase; range, 0.2 to 37; 95% CI, 1.4 to 4.4) and BFU-E (2.5-fold mean increase; range, 0.4 to 15; 95% CI,
0.8 to 3.7) following administration of rhTPO and G-CSF; for all CFUs,
the largest number of colonies was observed on the days of apheresis
(day 5 and 6 after the last dose of rhTPO). rhTPO dose and schedule had
no significant effect on the rate of CFU formation from baseline to
postmobilization. There was no statistically significant trend favoring
any particular rhTPO dosing schedule when we assessed the amount of
CFU-MK, BFU-E, CFU-GEMM, and CFU-GM in the stem-cell products, although
the highest yields were seen following sequential administration of
rhTPO 0.6 µg/kg, as illustrated in Table 6.
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Table 6.
CFU-GM, CFU-GEMM, CFU-MK and BFU-E Growth by rhTPO Dose
in Patients Mobilized by rhTPO in Combination With G-CSF
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Toxicities.
We observed no allergic reactions associated with the administration of
rhTPO. The superior vena cava occlusion in one patient assigned to
receive a single injection of rhTPO 0.6 µg/kg preceded the
administration of rhTPO and was most likely due to a previous catheter
infection; her platelet counts remained within the normal range (234 × 109/L and 151 × 109/L)
on the days of apheresis. She underwent high-dose therapy 6 months
later without any thromboembolic complications.
We observed platelet count elevations greater than 600 × 109/L lasting for 24 hours in two of six patients (peaks of
995 × 109/L and 964 × 109/L)
treated with rhTPO 0.6 µg/kg on days 3, 1, and 1 (arm
B). Both peaks occurred on the first day of apheresis, and platelet counts decreased by the second day of apheresis. To assess further the
possibility of excessive thrombocytosis following repeated administration, an additional rhTPO dose level was added, and six
patients received three doses of rhTPO 0.3 µg/kg every other day.
Platelet counts in these patients never reached 600 × 109/L.
Serially tested serum prior and subsequent to
the administration of rhTPO and post-high-dose chemotherapy remained
negative for the presence of any neutralizing antibodies in all subjects.
| |
DISCUSSION |
C-Mpl ligand, by expanding, and possibly by protecting, the
progenitor-cell pools of megakaryocytic, myelomonocytic, and erythroid lineage, can prevent or ameliorate hematopoietic toxicity inflicted by
cytotoxic therapy.15-17 Thrombopoietin may also accelerate
hematopoietic recovery following chemotherapy. When administered after
carboplatin and cyclophosphamide a truncated, pegylated form of the
C-Mpl ligand, PEG-rHuMGDF (MGDF) enhanced platelet recovery in a
dose-dependent fashion when given together with G-CSF.
CD34+ cell subsets, in patients treated at higher doses of
MGDF (0.3 to 5 µg/kg), were increased and platelet counts increased
to greater than 1,000,000/µL in 11 of 31 patients.22 MGDF
(0.03 to 5 µg/kg), when administered after carboplatin and
paclitaxel, resulted in less pronounced and shorter median platelet
nadirs compared with controls.23 Similarly, single
injections of rhTPO can attenuate thrombocytopenia when administered
before high-dose carboplatin; rhTPO 1.2 µg/kg given after high-dose
carboplatin may prevent the need for platelet
transfusions.24
Both the full-length and pegylated, truncated molecules are capable of
expanding the progenitor pool. rhTPO increased the number of
multilineage progenitors in the bone marrow while mobilizing CD34+ cells into the peripheral blood in cancer patients.
MGDF in combination with a variety of growth factors has been shown to
expand the number of megakaryocyte progenitors ex
vivo.25,26
We have found a substantial increase in the CD34+
progenitor-cell content of apheresis products when rhTPO, in
combination with G-CSF, was administered several days before apheresis.
While one can argue that prolonged administration of 10 µg/kg G-CSF alone for up to 9 days (rather than 3 days) before initiating apheresis
may increase the CD34+ yield, there are data to the
contrary. In a study conducted in normal volunteers, administration of
10 µg/kg G-CSF resulted in peak peripheral blood CD34+
cell counts on day 4 in two of five healthy subjects, while a broader
distribution of multiple peak CD34+ cell counts (between
days 3 and 7) was observed in three other volunteers.27
Since the peripheral blood CD34+ cell count predicts for
progenitor-cell yield in the apheresis product, we applied such short
G-CSF course before apheresis in our 20 control patients. Indeed, the
highest CD34+ yields were procured during the first and
second apheresis in 65% of patients; yields in the other 35% of
patients were highest between days 6 and 8. No patient in the control
group yielded 3 × 106 CD34+ cells/kg
beyond the sixth day of administering G-CSF, making it unlikely that
prolonged administration of G-CSF would lead to a decrease in the
number of aphereses needed to reach our target.
Patients in the rhTPO- and G-CSF-mobilized group and the
control group were comparable in their exposure to the number of prior
chemotherapy regimens and cycles; there were, however, half as many
patients in the rhTPO-treated cohort with a history of prior radiation
exposure (15% v 30%). Since we procured the targeted numbers
of CD34+ cells/kg in 61% of patients in the
rhTPO-mobilized group versus in 10% of controls with only one
apheresis procedure, it is likely that this sixfold difference was the
consequence of exposure to rhTPO and not due to any pretreatment
imbalance between the study cohort and controls. Indeed, when we
analyzed our data after stratifying for exposure for prior radiation
therapy, the beneficial effect of rhTPO on procurement and engraftment
had been confirmed. Although the number of patients with prior
radiation exposure was small, a trend suggesting favorable effects of
rhTPO on CD34+ cell yield and hematopoietic recovery was
observed in all groups. Should our findings hold true in larger cohorts
of patients (assuming that the CD34+ yield can be
calculated expeditiously) the majority of high-dose chemotherapy
candidates would require only a single apheresis procedure following
rhTPO/G-CSF mobilization.
A large increase in the numbers of CFU-GEMM, CFU-GM, CFU-MK, and BFU-E
over baseline was seen in the peripheral blood 5 to 6 days following
the last dose of rhTPO. The observed rise in peripheral blood platelet
concentration during the same period was probably due to both
accelerated proliferation/increased mitotic activity and terminal
differentiation since receptors for thrombopoietin are present on the
surface of both megakaryocytes and platelets.28-30
Although a single injection of rhTPO or MGDF effectively stimulated
platelet production,16 we have found that sequential administration of rhTPO at 0.6 µg/kg for three doses resulted in the
highest CD34+ cell yield and peripheral blood platelet
concentration and generated the highest percentage of cells of the
megakaryocyte lineage as documented by CD41+ marking within
the CD34+ progenitor-cell subset and by measuring CFU-MK
growth. This schedule also produced the highest PBPC platelet content.
While these findings need further confirmation in larger numbers of
patients, repeated stimulation of the rhTPO receptor (C-Mpl) may be the
most efficient way to expand both multipotential and megakaryocytic
progenitor-cell pools in addition to generating the largest number of
platelets in the peripheral blood.31
Accelerated recovery of the erythroid lineage and decreased erythrocyte
and platelet transfusion requirements in comparison to our control
group, which was closely matched for clinical and pretreatment
characteristics, suggested a clinically broader benefit of rhTPO in
patients undergoing high-dose chemotherapy.32-35 The observed decrease in erythrocyte transfusion requirements are likely to
be due to stimulatory effects of rhTPO on stem and early progenitor
cells, although it is possible that these effects are due to partial
sequence homology between thrombopoietin and
erythropoietin.8 Enhanced CFU-MK formation and an increase
in the number of CD41+ cells within the CD34+
population possibly accelerated differentiation. The resulting increase
in peripheral blood platelet concentration and platelet yield of PBPC
products might have helped to reduce platelet transfusion requirements;
indeed, one patient in the rhTPO-mobilized group required no platelet transfusions.
rhTPO can be used to generate large numbers of both progenitor cells
and platelets. Investigators have tested the effects of MGDF in healthy
donors and found substantially increased platelet yields in the
platelet apheresis products.36,37 We observed a strong
correlation between peripheral blood platelet concentration and
platelet content of PBPC products; hence, confirmation of our most
effective sequential schedule of delivery has clinical relevance.
However, one has to be careful not to overstimulate CFU-MK production
so as to avoid persistently elevated peripheral blood platelet counts.
Platelet and PBPC apheresis procedures need to be timed based on
careful daily monitoring of peripheral blood platelet counts to
optimize platelet and PBPC yield and prevent iatrogenic thrombocytosis.
In addition, although we have not seen development of neutralizing
antibodies and secondary thrombocytopenia in any of our patients,
vigilant follow-up evaluation is necessary.
In summary, administration of rhTPO in combination with G-CSF was safe
and well tolerated. rhTPO mobilization in combination with G-CSF
increased the CD34+ progenitor-cell and mononuclear yield,
peripheral blood platelet count, and platelet content of PBPC products.
We observed an increase in CFU-MK, CFU-GEMM, CFU-GM, and BFU-E
formation and platelet generation 5 to 6 days following the last dose
of rhTPO compared to baseline. Of the doses tested, rhTPO 0.6 µg/kg
given three times every other day produced the highest
CD34+ progenitor yield, platelet count, and the highest
percentage of CD41+ cells within the CD34+
progenitor-cell subset; the largest numbers of CFU-MK, CFU-GM, CFU-GEMM, and BFU-E were also associated with this schedule. rhTPO in
combination with G-CSF decreased the number of aphereses and has the
potential to necessitate only a single apheresis procedure in the
majority of patients. rhTPO accelerated granulocyte and platelet
recovery, and decreased erythrocyte transfusion requirements in breast
cancer patients undergoing high-dose chemotherapy with stem-cell
support versus a comparable and identically treated nonrandomized
control group. A prospective, randomized study to further clarify the
optimal dose, schedule, and benefits resulting from the administration
of rhTPO and G-CSF versus G-CSF alone is planned.
| |
NOTE ADDED IN PROOF |
The most active dose and schedule of rhTPO and G-CSF that were
identified in our study have been tested in a randomized,
placebo-controlled, phase II study. Preliminary results suggest that
this regimen has significant activity in mobilizing PBPC compared with
G-CSF alone.38
| |
ACKNOWLEDGMENT |
We thank Judy Brent, Debra Rader, and Debbie Reardon for their
assistance with the clinical aspects and data management of this study,
Barbara Nowicki for the flow cytometry analysis, and Yolanda Tamayo and
Melinda Kirk for secretarial assistance.
| |
FOOTNOTES |
Submitted August 14, 1998; accepted December 18, 1998.
Supported by National Cancer Institute Grants No. CA33572, CA62505, and
CA63265, Genentech Inc, and the Mrs Adelyn Luther and the Ladish Family
Foundations, and the Levien Foundation.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to George Somlo, MD, City of Hope National
Medical Center, 1500 E Duarte Rd, Duarte, CA 91010-3000.
| |
REFERENCES |
1.
de Sauvage FJ, Hass PE, Spencer SD, Malloy BE, Gurney AL, Spencer SA, darbonne WC, Henzel WJ, Wong SC, Kuang W-J, Oles KJ, Hultgren B, Solberg LA Jr, Goeddel DV, Eaton DL:
Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand.
Nature
369:533, 1994[Medline]
[Order article via Infotrieve]
2.
Bartley TD, Bogenberger J, Hunt P, Li YS, Lu HS, Martin F, Chang MS, Samal B, Nichol JL, Swift S, Johnson MJ, Hsu RY, Parker VP, Suggs S, Skrine JD:
Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl.
Cell
77:1117, 1994[Medline]
[Order article via Infotrieve]
3.
Lok S, Kaushansky K, Holly RD, Kuijper JL, Lofton-Day CE, Oort PJ, Grant FJ, Heipel MD, Burhead SK, Kramer JM, Bell LA, Sprecher CA, Blumberg H, Johnson R, Prunkard D:
Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo.
Nature
369:565, 1994[Medline]
[Order article via Infotrieve]
4.
Kaushansky K, Lok S, Holly RD, Broudy VC, Lin N, Bailey MC, Forstrom JW, Buddle MM, Oort PJ, Hagen FS, Roth GJ, Papayannopoulou T, Foster DC:
Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin.
Nature
369:568, 1994[Medline]
[Order article via Infotrieve]
5.
Wendling F, Maraskovsky E, Debili N, Florindo C, Teepe M, Titeux M, Methia N, Breton-Gorius J, Cosman D, Vainchenker W:
c-Mpl is a humoral regulator of megakaryocytopoiesis.
Nature
369:571, 1994[Medline]
[Order article via Infotrieve]
6.
Kaushansky K:
Thrombopoeitin: The primary regulator of platelet production.
Blood
86:419, 1995[Free Full Text]
7.
Young JC, Bruno E, Luens KM, Wu S, Backer M, Murray LJ:
Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+thy-1+Lin- primitive progenitor cells.
Blood
88:1619, 1996[Abstract/Free Full Text]
8.
Kaushansky K, Broudy VC, Grossman A, Humes J, Lin N, Ren HP, Bailey MC, Papayannopoulou, Forstrom JW, Sprugel KH:
Thrombopoietin expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelosuppressive therapy.
J Clin Invest
96:1683, 1995
9.
Frei E III, Antman K, Teicher B, Eder P, Schnipper L:
Bone marrow autotransplantion for solid tumor-prospects.
J Clin Oncol
11:515, 1989
10.
Bezwoda WR, Seymour L, Dansy RD:
High-dose chemotherapy with hematopoietic rescue as primary treatment for metastatic breast cancer: A randomized trial.
J Clin Oncol
13:2483, 1995[Abstract]
11.
Nademanee A, Sniecinski I, Schmidt GM, Dagis AC, O'Donnell MR, Snyder DS, Parker PM, Stein AS, Smith EP, Molina A, Stepan DE, Somlo G, Margolin KA, Woo D, Niland JC, Forman SJ:
High-dose chemotherapy followed by autologous peripheral blood transplantation for patients with Hodgkin's disease and non-Hodgkin's lymphoma using unprimed and colony-stimulating factor-mobilized peripheral-blood stem cells.
J Clin Oncol
12:2176, 1994[Abstract/Free Full Text]
12.
Klumpp TR, Mangan KF, Goldberg SL, Pearlman ES, Macdonald JS:
Granulocyte colon-stimulating factor accelerates neutrophil engraftment following peripheral-blood stem-cell transplantation: A prospective, randomized trial.
J Clin Oncol
13:1323, 1995[Abstract]
13.
Rebulla P, Finazzi G, Marangoni F, Avvisati G, Gugliotta L, Tognoni G, Barbui T, Mandelli F, Sirchia G:
The threshold for prophylactic platelet transfusions in adults with acute myeloid leukemia.
N Engl J Med
337:1870, 1997[Abstract/Free Full Text]
14.
The trial to reduce alloimmunization to platelets study group:
Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions.
N Engl J Med
337:1861, 1997[Abstract/Free Full Text]
15.
Neelis KJ, CC Hartong S, Egeland T, Thomas GR, Eaton DL, Wagemaker G:
The efficacy of single-dose administration of thrombopoietin with co-administration of either granulocyte/macrophage or granuloycte colony-stimulating factor in myelosuppressed rhesus monkeys.
Blood
90:2565, 1997[Abstract/Free Full Text]
16.
Ohwada A, Rafii S, AS Moore M, Cyrstal RG:
In vivo adenovirus vector-mediated transfer of the human thrombopoietin cDNA maintains platelet levels during radiation- and chemotherapy-induced bone marrow suppression.
Blood
88:778, 1996[Abstract/Free Full Text]
17.
Vadhan-Raj S, Murray LJ, Bueso-Ramos C, Patel S, Reddy SP, Hoots WK, Johnston T, Papadopolous NE, Hittelman WN, Johnston DA, Yang TA, Paton VE, Cohen RL, Hellmann SD, Benjamin RS, Broxmeyer HE:
Stimulation of megakaryocyte and platelet production by a single dose of recombinant human thrombopoietin in patients with cancer.
Ann Intern Med
126:673, 1997[Abstract/Free Full Text]
18.
Somlo G, Sniecinksi I, Odom-Maryon T, Nowicki B, Chow W, Hamasaki V, Leong L, Margolin K, Morgan R Jr., Raschko J, Shibata S, Tetef M, Molina A, Berenson RJ, Forman SJ, Doroshow JH:
Effect of CD34+ selection and various schedules of stem cell reinfusion and granuloycte colony-stimulating factor priming of hematopoietic recovery after high-dose chemotherapy for breast cancer.
Blood
89:1521, 1997[Abstract/Free Full Text]
19.
Somlo G, Doroshow JH, Forman SJ, Odom-Maryon T, Lee J, Chow W, Hamasaki V, Leong L, Morgan R Jr., Margolin K, Raschko J, Shibata S, Tetef M, Yen Y, Simpson J, Molina A:
High-Dose chemotherapy and stem-cell rescue in the treatment of high-risk breast cancer: Prognostic indicators of progression-free and overall survival.
J Clin Oncol
15:2882, 1997[Abstract]
20.
Lehmann EL:
Nonparametrics: Statistical Methods Based on Ranks. San Francisco, CA, Holden-Day, 1975.
21.
Toernqvist L, Pentti Vartia P, Yrjoe O, Vartia YO:
How should relative changes be measured?
Am Stat
39:43, 1985
22.
Basser RL, Rasko JEJ, Clarke K, Cebon J, Green MD, Grigg AP, Zalcberg J, Cohen B, O'Byrne J, Menchaca DM, Fox RM, Begley CG:
Randomized, blinded, placebo-controlled phase I trial of pegylated recombinant human megakaryocyte growth and development factor with filgrastim after dose-intensive chemotherapy in patients with advanced cancer.
Blood
89:3118, 1997[Abstract/Free Full Text]
23.
Fanucchi M, Glaspy J, Crawford J, Garst J, Figlin R, Sheridan W, Menchaca D, Tomita D, Ozer H, Harker L:
Effects of polyethylene glycol-conjugated recombinant human megakaryocyte growth and development factor on platelet counts after chemotherapy for lung cancer.
N Engl J Med
336:404, 1997[Abstract/Free Full Text]
24.
Vadhan-Raj S, Verschraegen C, McGarry L, BuesoRamos C, Kudelaka A, Freedman R, Edwards C, Bevers M, Levenback C, Gershenson D, Jones D, Yang T, Bast RC, Kavanagh J:
Recombinant human thrombopoietin (rhTPO) attenuates high-dose carboplatin (C)-induced thrombocytopenia in patients with gynecological malignancies.
Blood
90:580a, 1997 (suppl 1, abstr)
25.
Bertolini F, Battaglia M, Pedrazzoli P, Da Prada GA, Lanza A, Soligo D, Caneva L, Sarina B, Murphy S, Thomas T, della Cuna GR:
Megakaryocytic progenitors can be generated ex vivo and safely administered to autologous peripheral blood progenitor cell transplant recipients.
Blood
89:2679, 1997[Abstract/Free Full Text]
26.
Murray LJ, Luens KM, Estradad MF, Bruno E, Hoffman R, Cohen RL, Ashby MA, Vadhan-Raj S:
Thrombopoietin mobilizes CD34+ cell subsets into peripheral blood and expands multilineage progenitors in bone marrow of cancer patient with normal hematopoiesis.
Exp Hematol
26:207, 1998[Medline]
[Order article via Infotrieve]
27.
Grigg AP, Roberts AW, Raunow H, Houghton S, Layton JE, Boyd AW, McGrath KM, Maher D:
Optimizing dose and scheduling of filgastrim (granulocyte colony-stimulating factor) for mobilization and collection of peripheral blood progenitor cells in normal volunteers.
Blood
86:4437, 1995[Abstract/Free Full Text]
28.
Cramer EM, Norol F, Guichard J, Breton-Gorius JM, Vainchenker W, Masse J-M, Debili N:
Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand.
Blood
89:2336, 1997[Abstract/Free Full Text]
29.
Arnold JT, Daw NC, Stenberg PE, Jayawardene D, Srivastava DK, Jackson CW:
A single injection of pegylated murine megakaryocyte growth and development factor (MGDF) into mice is sufficient to produce a profound stimulation of megakaryocyte frequency, size, and ploidization.
Blood
89:823, 1997[Abstract/Free Full Text]
30.
Norol F, Vitrat N, Cramer E, Guichard J, Burstein SA, Vainchenker W, Debili N:
Effects of cytokines on platelet production from blood and marrow CD34+ cells.
Blood
91:830, 1988[Abstract/Free Full Text]
31.
Broudy VC, Lin NL, Sabath DF, Papayannopoulou T, Kaushansky K:
Human platelets display high-affinity receptors for thrombopoietin.
Blood
89:1896, 1997[Abstract/Free Full Text]
32.
Chao NJ, Schriber JR, Grimes K, Long GD, Negrin RS, Raimondi CM, Horning SJ, Brown SL, Miller L, Blume KG:
Granulocyte colony-stimulating factor "mobilized" peripheral blood progenitor cells accelerate granulocyte and platelet recovery after high-dose chemotherapy.
Blood
81:2031, 1993[Abstract/Free Full Text]
33.
Bensinger W, Singer J, Appelbaum F, Lilleby K, Longin K, Rowley S, Clarke E, Clift R, Hansen J, Shields T, Storb R, Weaver C, Weiden P, Buckner CD:
Autologous transplantation with peripheral blood mononuclear cells collected after administration of recombinant granulocyte stimulating factor.
Blood
81:3158, 1993[Abstract/Free Full Text]
34.
Debili N, Wendling F, Cosman D, Titeux M, Florindo C, Dusanter-Fourt I, Schooley K, Methia N, Charon M, Nador R, Bettaieb A, Vainchenker W:
The Mpl receptor is expressed in the megakaryocytic lineage from late progenitors to platelets.
Blood
85:391, 1995[Abstract/Free Full Text]
35.
Bensinger W, Appelbaum F, Rowley S, Storb R, Sanders J, Lilleby K, Gooley t, Demirer T, Schiffman K, Weaver C, Clift R, Chauncey T, Klarnet J, Montgomery P, Petersdorf S, Weiden P, Witherspoon R, Buckner CD:
Factors that influence collection and engraftment of autologous peripheral-blood stem cells.
J Clin Oncol
13:2547, 1995[Abstract]
36.
Goodnough LT, DiPersio J, McCullough J, et al:
Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) increases platelet count and apheresis yields in normal donors.
Transfusion
37:67S, 1997
37.
Tomita D, Petrarca M, Paine T, Olson K, Roskos I, Cheung E:
Effect of single dose of pegylated human recombinant megakaryocyte growth factor (PEG-rHuMGDF) on platelet counts: Implications for platelet apheresis.
Transfusion
37:2S, 1997
38.
Linker C, Anderlini P, Herzig R, Somlo G, Christiansen N, Fay J, Lynch J, Bensinger W, Goodnough T, Benyunes M, Ashby M, Jones D, Weaver C:
A randomized, placebo-controlled, phase II trial of recombinant human thrombopoietin (rhTPO) in subjects undergoing high-dose chemotherapy (HDC) and PBPC transplant.
Blood
92:682a, 1998 (abstr, suppl 1)
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ABSTRACT
INTRODUCTION
