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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1532-1540
A Randomized Phase-II Study of BB-10010 (Macrophage Inflammatory
Protein- 1 ) in Patients With Advanced Breast Cancer Receiving
5-Fluorouracil, Adriamycin, and Cyclophosphamide Chemotherapy
By
Mark J. Clemons,
Ernest Marshall,
Jan Dürig,
Ken Watanabe,
Anthony Howell,
David Miles,
Helena Earl,
Julie Kiernan,
Audrey Griffiths,
K. Towlson,
P. DeTakats,
Nydia G. Testa,
Mark Dougal,
Michael G. Hunter,
L. Michael Wood,
Lloyd G. Czaplewski,
Andrew Millar,
T. Michael Dexter, and
Brian I. Lord
From the CRC Department of Medical Oncology and Paterson Institute
for Cancer Research, Christie Hospital, Manchester; ICRF Clinical
Oncology Unit, Guy's Hospital, London; CRC Institute of Cancer
Studies, Birmingham; Department of Medical Statistics, Christie
Hospital, Manchester; and British Biotech plc, Oxford, UK.
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ABSTRACT |
BB-10010 is a variant of the human form of macrophage inflammatory
protein-1 (MIP-1), which has been shown in mice to block the
entry of hematopoietic stem cells into S-phase and to increase their
self-renewal capacity during recovery from cytotoxic damage. Its use
may constitute a novel approach for protecting the quality of the stem
cell population and its capacity to regenerate after periods of
cytotoxic treatment. Thirty patients with locally advanced or
metastatic breast cancer were entered into the first randomized, parallel group controlled phase II study. This was designed to evaluate
the potential myeloprotective effects of a 7-day regimen of BB-10010
administered to patients receiving six cycles of 5-fluorouracil (5-FU),
adriamycin, and cyclophosphamide (FAC) chemotherapy. Patients were
randomized, 10 receiving 100 µg/kg BB-10010, 11 receiving 30 µg/kg
BB-10010, and nine control patients receiving no BB-10010. BB-10010 was
well-tolerated in all patients with no severe adverse events related to
the drug. Episodes of febrile neutropenia complicated only 4% of the
treatment cycles and there was no difference in incidence between the
treated and nontreated groups. Studies to assess the generation of
progenitor cells in long-term bone marrow cultures were performed
immediately preceding chemotherapy and at the end of six dosing cycles
in 18 patients. Circulating neutrophils, platelets, CD
34+ cells, and granulocyte/macrophage colony-forming cell
(GM-CFC) levels were determined at serial time points in
cycles 1, 3, and 6. The results showed similar hemoglobin and platelet
kinetics in all three groups. On completion of the six treatment
cycles, the average pretreatment neutrophil levels were reduced from
5.3 to 1.7 × 109/L in the control patients and from 4.3 to 1.9 and 4.5 to 2.5 × 109/L in the 30/100 µg/kg
BB-10010 groups, respectively. Relative to their pretreatment values,
50% of the patients receiving BB-10010 completed the treatment with
neutrophil values significantly higher than any of the controls
(P = .02). Mobilization of GM-CFC was enhanced by BB-10010
with an additional fivefold increase over that generated by
chemotherapy alone, giving a maximal 25-fold increase over pretreatment
values. Bone marrow progenitor assays before and after this standard
regimen of chemotherapy indicated little long-term cumulative
impairment to recovery from chemotherapy. Despite the limited
cumulative damage to the bone marrow, which may have minimized the
protective value of BB-10010 during this regimen of chemotherapy,
better recovery of neutrophils in the later treatment cycles with
BB-10010 was indicated in a number of patients.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
DOXORUBICIN-BASED chemotherapy represents
the mainstay of treatment for patients with breast cancer, both in the
adjuvant and advanced setting. Dose escalation results in a higher
response rate, but it is also associated with increased hematopoietic
toxicity and dose-limiting mucosal toxicity.1,2 In the
adjuvant setting, repeated cycles of doxorubicin-based treatment have
been shown to produce cumulative and long-lasting damage in the bone
marrow (BM) progenitor cell populations.3 The effects of
short-term hematopoietic damage resulting from chemotherapy can be
overcome to some extent by the concurrent use of granulocyte
colony-stimulating factor (G-CSF) to encourage regeneration of
neutrophils.2,4 However, this approach is accompanied by
progressive thrombocytopenia and probable cumulative BM damage as
reflected by a reduction in the quality of mobilized progenitor cells
over successive cycles of treatment.5,6
Currently, there is considerable interest in chemotherapy dose
intensification as a means of improving tumor response rates and
perhaps survival.7-9 The introduction of G-CSF and other hematopoietic growth factors has facilitated this movement, but the
small gain (typically less than twofold) has been compounded by
increased hematopoietic toxicity with progressive
thrombocytopenia,5,10,11 probably as a consequence of
incremental damage to the BM stem cell pool.6,12 The
ability to maintain stem cells in a quiescent state during periods of
chemotherapy treatment in animals has been shown to attenuate the
cytotoxic effects on hematopoeisis13-20 and
epithelium,21 the former reflected in an accelerated BM and
peripheral blood cell recovery.
Macrophage inflammatory protein-1 (MIP-1), a C-C
chemokine22 has been recognized as a hematopoietic stem
cell proliferation inhibitor that enhances stem cell recovery,
postcytotoxic treatment, not only as a consequence of cytoprotection,
but also via secondary effects on stem cell self-renewal.23
BB-10010 is an active, nonaggregating variant of human
MIP-1.24 In mice, it reduced the degree of accumulated
hematopoietic damage after repeated sublethal
irradiations25 and, in a further model, enhanced leukocyte recovery and progenitor cell mobilization after
cyclophosphamide.26
In phase-I clinical studies, BB-10010 was extremely well-tolerated with
no maximum tolerated dose defined at up to 300 µg/kg administered
subcutaneously and up to 100 µ/kg
intravenously.27 Only a few mild local injection site
reactions were observed despite its earlier classification as a
proinflammatory chemokine22 and a single subcutaneous (SC)
injection resulted in sustained plasma levels over a 24-hour period.
We now report the effects of BB-10010 on the maintenance of
hematopoiesis in humans, using BM and peripheral blood parameters, following a repeated daily dosing schedule, administered with every
cycle of 5-fluorouracil, adriamycin, and cyclophosphamide (FAC)
chemotherapy in patients with advanced breast cancer.
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MATERIALS AND METHODS |
Between August, 1995 and March 1996, 30 patients with advanced breast
cancer were recruited from three centers: 20 at the Christie Hospital,
Manchester, 9 at Guy's Hospital, London, and 1 at the Institute of
Cancer Studies, Birmingham. The protocol was approved by the South
Manchester Ethical Committee and written informed consent was obtained
from all patients. Patient inclusion criteria included the following:
females aged 18 years or older with advanced breast cancer and Eastern
Cooperative Oncology Group (ECOG) performance status < 2. White cell count (WCC)  3 × 109/L, platelets 100 × 109/L, and hemoglobin (Hb) 11 g/dL. They had normal renal function tests, normal bilirubin, and
transaminases less than two times the upper limit of normal. Exclusion
criteria included previous chemotherapy, except standard adjuvant
cyclophosphamide, methotrexate, and fluorouracil (CMF)
chemotherapy at least 6 months previously and previous radiotherapy to
more than one third of the axial skeleton.
Randomization.
Patients were randomized to receive either 0, 30, or 100 µg
BB-10010/kg SC daily for 7 days (days  1, 0, 1, 2, 3, 4, and 5) administered at the start of each of the six cycles of FAC chemotherapy (day 0), which were given at 21-day intervals. Control patients received chemotherapy only. Subjects were balanced by the method of
minimization according to whether they had received previous CMF
adjuvant chemotherapy or not, plus the extent of any metastases in
their hematopoietic system. These were categorized as follows: (1)
locally advanced disease with a negative bone scan; (2) evidence of
metastatic disease in less than three marrow containing areas of
skeleton; and (3) evidence of metastatic disease in all three of the
principal marrow containing areas of the skeleton (ribs/pelvis/spine).
Cytotoxic chemotherapy.
All patients received intravenous FAC chemotherapy (5-FU 600 mg/m2 day 0, doxorubicin 50 mg/m2, and
cyclophosphamide 600 mg/m2) on day 0 and subsequently at
21-day intervals. In each cycle, the chemotherapy was administered 24 hours after the start of the BB-10010 dosing. All subjects received a
7-day course of prophylactic ciprofloxacin and fluconazole if their
neutrophil count dropped below 0.5 × 109/L at any
time during the study. If neutrophils or platelets had not recovered to
1 × 109/L or 100 × 109/L,
respectively, chemotherapy was to be delayed by up to 2 weeks. Dose
reductions of 25% were to be made in succeeding cycles in event of
either (1) an episode of neutropenic sepsis (temperature 38°C
and neutrophils < 0.5 × 109/L); (2) any
nonhematopoietic National Cancer Institute (NCI) toxicity
of grade 3 or 4; or (3) failure of the neutrophil count to recover
following a 1-week delay.
BB-10010.
BB-10010 was supplied by British Biotech Pharmaceuticals Ltd as a
sterile solution at 2 mg/mL concentration in phosphate-buffered saline
(PBS). Ampules of drug were stored at 20°C then thawed within 7 days before administration and stored at 4°C. The correct volume of BB-10010 was drawn into a 25-gauge 1 inch sterile needle from
either the 10 mg/mL ampule (for the 100 µg/kg dose level) or the 2 mg/mL ampule (for the 30 µg/kg dose level). The BB-10010 was
administered as a daily (7 days) SC injection, the first dose being
given 24 hours before each cycle of FAC chemotherapy.
Peripheral blood assays.
Full blood counts (FBC) were taken at screening and on days 7, 10, 14, 16, and 20 of cycles 1, 3, and 6 plus day 20 of cycles 2, 4, and 5. Circulating progenitor cells were assayed on days 14, 16, and 20 of
cycles 1, 3, and 6, in 11 of the 20 patients attending the Manchester
center, using a clonogenic assay for GM-CFC. CD34+ cells
were assessed by flow cytometry.
GM-CFC assay.
At the time points specified above, mononuclear cells (MNC) from
peripheral blood were separated on Ficoll Hypaque (density 1.077 g/mL;
Life Technologies, Paisley, UK) and suspended in Iscove's medium
supplemented with 4 × 10 3 mol/L glutamine,
107 mol/L sodium selenite, 2.5 × 104 mol/L thioglycerol, 30% pretested fetal
calf serum (FCS), 1% deionized bovine serum albumin (BSA) (Sigma
Chemical Co, Poole, UK), and 2 U of recombinant erythropoietin (Epo)
(Boehringer Mannheim, Lewis, UK) per mL of culture. Medium conditioned
by the cell line 5637 was added at 10% by volume as source of growth
factors. Cells were cultured in 1.35% methylcellulose in 24-well
standard tissue culture plates (Falcon; Fred Baker Supplies, Runcorn,
Cheshire, UK) in triplicate, at a final concentration of
105 mononuclear cells/mL/well. The cells were incubated at
37°C in a humidified atmosphere of 5% C02 and 5%
O2 in nitrogen. Colonies were counted and classified after
14 days growth as granulocyte-macrophage colonies, as previously
described.28
Determination of CD34+ cells.
The number of CD34+ cells in the peripheral blood was
determined on days 14, 16, and 20 of cycles 1, 3, and 6. Fifty-microliter aliquots of the samples were incubated with a mouse
anti-CD34 fluorescein isothiocyanate (FITC)-conjugated monoclonal
antibody (MoAb) (anti-human progenitor cell antigen-2; Becton
Dickinson, San Jose, CA) for 30 minutes at 4°C. Red blood cells
were lysed by addition of 2 mL ammonium chloride solution (Ortho-mune
lysing Reagent; Ortho Diagnostic Systems, Inc, Raritan,
NJ). Cells were washed twice in PBS, stored in a fixative
of 1% formaldehyde solution, and analyzed on a fluorescence-activated
cell sorter (FACS). A nonspecific isotype matched FITC-conjugated MoAb
was used as a negative control.29
BM assays.
A total of 2 mL of BM was aspirated under aseptic conditions from the
right posterior iliac crest of the Manchester patients before starting
treatment and at day 28 after the start of the sixth cycle of FAC
chemotherapy. Mononuclear cells were separated and assayed for GM-CFC
and CD34+ cells as described above. The pretreatment marrow
samples were also assessed for tumor infiltration, GM-CFC, and
CD34+ cells. Long-term BM cultures (LTBMC) were established
from both pretreatment and posttreatment samples.
LTBMC.
Between 5 × 105 and 2 × 107
nucleated BM cells after separation from red blood cells using
methylcellulose28 in 10 mL of LTBMC medium was added to 25 cm2 tissue culture flasks (Falcon). Each 10 mL of LTBMC
medium consisted of 1 mL FCS, 1 mL horse serum, 7.9 mL of Iscove's
modified Dulbecco's medium (IMDM) (350 mOsm/kg) and 0.1 mL of 5 × 10-5 mol/L
hydrocortisone succinate (Sigma). After inoculation, the flasks were
gassed with 5% carbon dioxide in air, then capped, and incubated at
33°C in the dark. Cultures were fed routinely on a weekly basis by
replacing half the volume of supernatant with fresh cell-free medium.
The cells in the harvested medium were assayed for GM-CFC. At 4 and 8 weeks of culture, some cultures were sacrificed for assay of GM-CFC in
the adherent layer after trypsin digestion as described by Couthino et
al.28
Statistical methods.
To assess differences between the treatment groups with respect to
timing and depth of neutrophil nadir, stabilization of counts during
the first 7 days of each cycle (corresponding with BB-10010 dosing) and
also recovery in counts at day 20 across each of the three cycles, the
absolute neutrophil counts were subjected to repeated measures analysis
of variance (ANOVA) multivariate method using Wilk's  test.
The magnitude of GM-CFC and total nucleated cell generation in pre- and
posttreatment LTBMCs were expressed by the area-under-the-curve (AUC)
over 8 weeks. The Kruskal-Wallis test and analysis of covariance were
used to compare the patient groups (control, BB-10010, 30 and 100 µg/kg) and differences between pre- and posttreatment values within
the groups. Repeated measures of covariance were performed to identify
differences in peripheral blood progenitor cell mobilization.
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RESULTS |
Patient characteristics are shown in Table
1. Their mean ages (and range) were 45 (29 to 63), 49 (34 to 62), and
56 (41 to 69) for the control (no BB-10010) and the 30 and 100 µg/kg
BB-10010 groups, respectively. The groups were well-matched for number of sites of disease and previous treatment.
All patients tolerated the FAC chemotherapy treatment extremely well
with a low incidence of neutropenic sepsis (4% of cycles, duration 3 to 6 days) in all groups. Three control patients were withdrawn, two
due to excessive disease progression and one withdrawn (and excluded
from Table 1) for surgery after a good response to chemotherapy. One
patient receiving 30 mg/kg BB-10010 was also withdrawn due to excessive
disease progression. Of the patients with neutropenic sepsis, 1 was
from the control group, 2 received 30 µg/kg BB-10010, 1 required a
single 1-week dose delay, and 1 required a 2-week delay; 2 patients
received 100 µg/kg BB-10010; 1 required a 1-week delay and the other
required a 2-week dose delay. Two of the four BB-10010 patients
requiring dose delays showed extremely poor neutrophil recovery even in
the first cycle of chemotherapy (see below). However, these delays have
been ignored in the following analyses because they were minimal (2%
of the total treatment cycles) and distributed at a low level among all three patient groups. Nonhematopoietic toxicity was typically mild with
no episodes of oral mucositis 2 NCI grade. Nausea was well-controlled
with standard antiemetics. BB-10010 administration was well-tolerated
and was not associated with any serious adverse events. Three patients
(one at 30 µg/kg and two at 100 µg/kg BB-10010) suffered a mild
self-limiting cutaneous reaction characterized by erythema and swelling
at the injection site. No patient developed any systemic inflammatory
response or anaphylaxis.
Pharmacokinetics of BB-10010.
Plasma concentrations of BB-10010 in blood samples taken on days
1, 0, 2, and 6 of the first cycle of FAC treatment were assayed,
under contract, at Huntingdon Life Sciences (Huntingdon, UK), by
hMIP-1 enzyme-linked immunosorbent assay (ELISA) (R & D Systems,
hMIP-1 Quantikine Kit; Abingdon, UK). This ELISA measures hMIP-1
and BB-10010 equally well and, as in the Phase I trial,27 no endogenous MIP-1 was detected in the controls: all were below the
detection limit of 93.6 pg/mL. At 30 µg/kg, 33 assays over all time
points gave an average concentration of 258 ± 146 pg/mL including
two, which were below the detection limit. At 100 µg/kg, 41 assays
gave an average concentration of 1,604 ± 2,050 pg/mL, including one
assay below the limit of detection. The large standard deviation was
the effect of two individual measurements that gave readings of 3.4 and
8 times the average for that group. The reasons for this variability
are not clear, but the overall excess levels of circulating BB-10010
over the whole injection schedule indicate its continuous availability,
and both dose levels used were therefore considered adequate for
effective dosing in the BM.27
Peripheral blood.
All patients, irrespective of their treatment group, retained good
levels of Hb and platelets throughout the period of treatment, both
parameters remaining well within their normal range and variations (Table 2).
Neutrophils.
The mean and standard deviations of the neutrophil counts, pretreatment
and at the ends of cycles 1, 3, and 6, are shown in Table 2 and for the
complete picture, as a percentage of their pretreatment levels in
Fig 1. Pretreatment values were 20% lower in the BB-10010 groups than in the control group. However, the difference was statistically not significant. Each cycle of
chemotherapy caused a dramatic reduction of the neutrophil counts with
nadirs, generally lasting from days 10 to 16 of each cycle, of 0.4 to 1.3 × 109/L followed by recovery by day 20. Table 3 charts the recovery pattern
relative to the starting point in each cycle. From this (and Fig 1), it
can be seen that after an initial fall to about 71% of pretreatment
values in all groups at the end of cycle 1, the second cycles showed no
further net damage; 97% recovery in the controls and 91% and 100%
recovery in the two BB-10010-treated groups. Thereafter, the control
group showed a progressive decline (92%, 76%, 65%) in its recoveries
over cycles 3 to 6, resulting in an overall reduction to 32% at the
end of six cycles of FAC chemotherapy. By contrast, little additional
damage occurred in the two treatment groups with 113%, 79%, and 82%
recoveries in cycles 3 to 6 and ending at 50% of pretreatment
neutrophil levels. It should be noted, however, that of the control
group, no patient exceeded 43% of her pretreatment neutrophil count at
day 20 of cycle 6. By contrast, of the 10 patients receiving 30 µg/kg
BB-10010, four repeatedly failed to attain 2 × 109
neutrophils/L (40% pretreatment value) even in the first treatment cycle and a further two also failed to surpass the maximal level (43%
of their pretreatment value) seen in the control cohort at the end of
six cycles. The remaining four in this group completed the trial with
higher neutrophil counts than the best of the controls. Similarly, in
the 100 µg/kg BB-10010 treated group, 6 of 10 patients completed the
trial with higher neutrophil counts than the best of the controls.
Overall, therefore, 50% of the BB-10010-treated patients performed
better than the control patients and a variance ratio test on the
standard deviations of these two groups showed them to be different
(P = .02).
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| Fig 1.
Mean neutrophil counts over cycles 1, 3, and 6 of FAC
chemotherapy, presented as a percentage of each patient's pretreatment
neutrophil count. Arrows on the abscissa indicate day 0 of the
treatment-cycle (C). ( ), control (no BB-10010); ( ), 30 µg/kg
BB-10010; ( ), 100 µg/kg BB-10010.
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Owing to the large standard deviations (interpatient variations),
however, few of these changes between cycles or between treatments
attained statistical significance. Repeated measures of ANOVA using
multivariate method was used to study the patterns of absolute
neutrophil counts over time and to relate them to the dose of BB-10010.
Of the 30 patients, three were omitted from the analysis, as they did
not complete the full six cycles of treatment. For the remaining 27 patients, the nadir days were averaged leaving 81 sets of measurements
over the three cycles (27 in cycle 1, 28 in cycle 3, and 26 in cycle
6). Although counts in the control group recovered from the
chemotherapy-induced nadirs by day 20 to levels that were statistically
equivalent to days 0 and 7 in each cycle, repeated measures analysis
used to study the change across cycles gave clear evidence of a
difference between cycles (Wilk's : P < .001). The
contrast between recoveries in cycles 1 and 3 was not significant
(P = .79), but that between cycles 1, 3, and 6 was highly
significant (P < .001). However, the difference
between means of the control and BB-10010 groups at the end of cycle 6, based on the raw data counts, was statistically not significant
(Wilk's : P = .2).
Progenitor and CD34+ cells.
Pretreatment, peripheral blood in the control patients contained 8 ± 3 (standard error [SE]) GM-CFC per mL and 1,100 ± 900 (SE) CD34+ cells per mL. Each cycle of FAC
treatment alone induced a modest mobilization of both cell types, over
days 16 to 20, to maximum levels of 53 and 6,500 per mL, respectively
(approximately up to sixfold). Additional BB-10010
treatment increased GM-CFC mobilization up to a maximum of 151 per mL,
and maximal levels of about 25 times the pretreatment level. A full
description of the levels of mobilization is shown in
Fig 2A and B. Although mobilization in the
sixth cycle was less striking, BB-10010 still increased GM-CFC levels
above those with FAC therapy alone (Fig 2A). CD34+ cells
were also mobilized in all cycles of treatment (again up to about
sixfold normal), but BB-10010 had little further impact (Fig 2B).
However, the considerable interpatient variability in the progenitor
and CD34+ cell assays meant there were no statistically
significant differences (P > .05) between the groups at any
individual time point. Nevertheless, GM-CFC were mobilized to a greater
extent in all of the nine sets of assays (Fig 2A) and the average
increase (4.7 ± 1.1-fold) may be seen as significant (P = .01).
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| Fig 2.
Mobilization kinetics of hematopoietic progenitor cells
through cycles C1, C3, and C6 of FAC chemotherapy. Data are presented
as a percentage of the pretreatment (PT) levels of GM-CFC (A) and of
CD34+ (B), (±SE bars). Control ( ) n = 4; BB-10010
() n = 7 at 30 plus 5 at 100 µg/kg.
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BM.
BM was sampled before the first treatment cycle and after the
completion of the last cycle. Both GM-CFC and CD34+ cells
were well maintained with recoveries after completion of the full
treatment schedules being close to their pretreatment levels
(Table 4).
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Table 4.
CD34+ and GM-CFC Content in Fresh BM
Harvested Before and After Administration of 6 Cycles of FAC
Chemotherapy
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Analysis of AUC to assess the numbers of either nucleated cells or
GM-CFC in the supernatant of LTBMC by the pre- and posttreatment marrows did not show any significant difference in the
BB-10010-treated patients compared with controls
(Fig 3). However, in cultures sacrificed at
4 weeks (though not at 8 weeks), posttreatment control patients had
about 10 times lower numbers of GM-CFC per culture adherent layer
compared with pretreatment (Table 5). Both
groups of BB-10010-treated patients, however, retained their
pretreatment levels (P = .04).
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| Fig 3.
Hematopoiesis over 8 weeks (GM-CFC and nucleated cells)
in LTBMCs established from patients before (A) and after completion of
six cycles of FAC chemotherapy (B). Symbol key: , control group
(n = 5); , BB-10010, 30 µg/kg (n = 7); ,
BB-10010, 100 µg/kg (n = 6). Values represent the mean ± SE.
Differences in the AUC between weeks 1 and 8 of culture for each
patient group, pretreatment and posttreatment, groups were not
statistically significant (P > .05).
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Table 5.
GM-CFC in the Adherent Layers of 4- and 8-Week Old LTBMC
Established Before and After Administration of 6 Cycles of FAC
Chemotherapy
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DISCUSSION |
This study reports on the potential myeloprotective effects of MIP-1
when administered during all cycles of treatment in a multicyclic
program of chemotherapy in a clinical trial. MIP-1, in a formulation
recognized as BB-10010,24 was administered at two dose
levels in combination with standard dose FAC chemotherapy. Its 7-day
injection schedule (starting 1 day before FAC therapy) was based on
preclinical models that had demonstrated superior efficacy of MIP-1
as a protracted administration.25,26 In this trial,
BB-10010 was administered at dose levels, which had been shown in
preliminary pharmacokinetic studies and a phase I healthy human
volunteer toxicity study, to be safe and to produce measurable plasma
levels of MIP-1.26 Its myelotoxic protective capacity
was assessed in terms of neutrophil recovery rate in each 21-day cycle
of FAC chemotherapy, mobilization of hematopoietic progenitor cells
into the peripheral blood, and the quality of progenitor cells in the
BM before and after six cycles of FAC treatment. The parallel
group-controlled design of this study, with BB-10010 administered in
every treatment cycle, allowed direct comparison of a protection arm
versus control (chemotherapy only). All patients tolerated the
chemotherapy well and only 5% of the treatment cycles were subject to
delays due to suboptimal recovery (primary center). There was no
mucosal toxicity and in keeping with our previous phase I studies,
BB-10010 itself was not associated with any significant toxicity,
although three patients developed mild recurring injection site
reactions characterized by erythema and weal formation.27 A
recently published Phase II study also found BB-10010 to be safe and
with no mucosal toxicity.
After chemotherapy, neutrophils in the blood underwent the anticipated
changes of decline to a very significant nadir and, with sequential
cycles of treatment, a reducing capacity to recover to pretreatment
levels by day 20 (Fig 1, Table 3). At no point were the results in the
BB-10010 treatment arms significantly different from those in the
control arm, however there was a high degree of interpatient variation
in all arms of this relatively small study. Furthermore, there were
also two patients in each of the two BB-10010 treatment arms who were
barely making the neutrophil recovery criterion between chemotherapy
cycles. The reasons for their failure are unclear, but seem unrelated
to their health status at the start of the study. All four
satisfactorily met the inclusion criteria and each had only one site of
secondary disease (Table 1). Two had breast/soft tissue involvement,
one had bone, and one had liver metastases. Therefore, they were not excluded from any of these analyses.
Because by chance, the average pretreatment neutrophil counts in both
the BB-10010 treatment arms were nearly 20% lower than in the control
arm, we have also presented the results as a percentage of the
pretreatment value, calculated as an average from each individual
patient. Other than the nadir days (days 10 to 16 of each cycle), the
neutrophil counts in the BB-10010-treated patients were generally
higher (Fig 1). Fall to the nadir appeared to be delayed (day 7) and
20-day neutrophil recoveries, particularly in the final treatment
cycle, were enhanced. There was even an indication of a BB-10010
dose-related response in the final treatment cycle. Because no obvious
protection was afforded by BB-10010 in the first treatment cycle, the
progressive protection over cycles 2 to 6 becomes more evident: the
63% subsequently accrued failure to recover fully in the controls was
reduced to only 23% by the treatment with BB-10010. At this stage,
despite being in a poorer group, 50% of the patients treated with
BB-10010 had neutrophil counts that were higher than those seen in any
of the controls (Fig 4). A recently
published phase II trial, limited to one cycle of cytotoxic treatment
only,30 was also unable to record any enhanced neutrophil
recovery due to BB-10010. The pattern of recovery now seen in the later
cycles, however, is in full accord with those seen in preclinical
studies with mice, where the full protective effects of BB-10010 were
seen only after several cycles of sublethal irradiation25
or bis-chloro-nitroso-urea treatment (unpublished data, E.M.).
Furthermore, it cannot be ascribed to any demargination phenomenon, as
phase I studies showed only a modest dose-related monocytophilia due to
BB-10010 and no effect on levels of circulating
neutrophils.27 This latter finding was also consistent with
the observation that human neutrophils are 10-fold less responsive to
BB-10010 than monocytes.31
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| Fig 4.
Histogram showing the distribution of neutrophil counts
(as percent of pretreatment value for each patient) at the end of the
study (cycle 6, day 20), comparing 20 patients receiving BB-10010 with
seven who did not. (), Control; (), BB-10010 treatments.
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Because MIP-1 was originally studied as a hematopoietic stem cell
protection factor, most of the in vivo, preclinical work in animals
relates to BM responses. In those published studies, which included
mature cells, there was some acceleration of neutrophil production as a
result.16,26 In studies using radiation and noncycle-specific cytotoxic drugs, long-term BM damage, damage that was
ameliorated by administration of BB-10010, accumulated in murine
hematopoietic spleen colony-forming units.25,26 Such effects were not seen, at least in the GM-CFC and CD34+
cell populations, in this study. Marrow harvested both before and after
completion of the six cycles of treatment contained a normal complement
of progenitor cells (Table 4). Furthermore, the marrows performed
equally well in generating these progenitor cells in long-term BM
cultures (Fig 3). BB-10010 did not influence these observations except
in maintaining some additional capacity for progenitor cell
mobilization into the peripheral blood. This was in agreement with
experimental findings in mice26,32 and consistent with the
preliminary findings of a phase I trial,33 which also
indicated about a fivefold increase relative to chemotherapy alone. The
extended time scale of release in these patients is, however, different
from that in mice, where BB-10010 induced short-term mobilization. The
reasons for this difference are not known. It is recognized, however,
that chemotherapy extends the mobilization process34 and it
is possible that the damage caused by therapy is also permissive for an
extended mobilizing effect of BB-10010. Perhaps surprisingly, the
additional mobilization was not reflected by CD34+ cells.
This population, however, is comprised of a much wider range of
hematopoietic cells and its larger complement may well have concealed
changes in the smaller GM-CFC component it contained. The only
deficiency in the quality of the BM after FAC treatment alone appeared
in its reduced capacity to establish the production of GM-CFC in the
adherent layers of long-term cultures within 4 weeks (Table 5). The
posttreatment kinetics of culture development were normal in marrow
from the BB-10010-treated patients, whereas the controls were slower
to establish their pretreatment performance. The different kinetics in
posttreatment BM harvests, together with the higher numbers of
mobilized progenitors seen in the BB-10010-treated patients (Fig 2),
may indicate somewhat more active marrows in post-BB-10010-treated
patients. The significant shortfall in the sixth cycle control
neutrophil recovery may well be a direct consequence of a less active
marrow in the control patients. The posttreatment marrows were
harvested 1 week after the completion of cycle 6 and the point at which
the final neutrophil counts were recorded. It is possible that this
delay would have allowed further recovery in the control neutrophils to
the levels recorded in the BB-10010-treated patients 1 week earlier.
Unfortunately, the appropriate kinetic studies on the BM, necessary to
resolve these questions, were not included in the trial design.
Nevertheless, it is clear that in these groups of patients, the
intensity of the FAC treatment regimen was insufficient to cause
significant long-term manifestation of accumulated myelotoxicty.
Compared with the preclinical animal experiments where cumulative
toxicity resulted in a reduction in BM spleen colony-forming cells of
about 65%, this was clearly insufficient to allow significant
manifestation of the potential of BB-10010 for myeloprotection.
In conclusion, therefore, it is clear that BB-10010 is well-tolerated
by the patients and, although basically considered to be an inhibitor
of hematopoiesis, does not cause additional myelosuppression. The
standard level of FAC therapy used was also well-tolerated and BB-10010
did not cause any significant changes in neutropenic sepsis. Although
there was no statistically significant benefit derived from BB-10010 in
this study, there was some evidence to suggest dose-dependent
improvements in neutrophil recovery at the end of cycles 3 and 6. The
ability to maintain acceptable levels of recovery in the face of
additional accumulating myelotoxicity due to high-dose cytotoxic
treatment or to an extended sequence of therapy cycles would allow the
potential for greater damage to the tumor(s) under treatment. On day 20 of cycle 6, the last data point in this study, we note that 50% of the
BB-10010-treated patients completed the trial with neutrophil counts
higher than those in the control group. BB-10010 did, therefore,
indicate a measure of myeloprotection against repeated cycles of FAC
chemotherapy. This suggested that additional cycles of treatment may
have demonstrated more fully the ability to maintain acceptable levels
of recovery in the face of additional accumulating myelotoxicity.
Further studies of additional cycles of therapy or using more intensive regimens of cytotoxicity, both designed to increase the degree of
accumulated myelotoxicity, will be necessary to establish whether this
apparent protection is real and to determine the efficiency and
therapeutic benefit of BB-10010.
| |
FOOTNOTES |
Submitted December 8, 1997;
accepted April 29, 1998.
Supported by the Cancer Research Campaign of Great Britain and British
Biotech Pharmaceuticals Ltd for whom Dr Ros Puttick coordinated the
trial and the Biometrics Department gave valuable advice. M.C. and E.M.
were supported by grants from The Leukaemia Research Fund (London, UK),
and J.D. is supported by a grant from the European Society for
Molecular Oncology (Brussels, Belgium).
Address reprint requests to Prof Anthony Howell, MD, CRC Department of
Medical Oncology, Christie Hospital NHS Trust, Manchester, M20 4BX, UK.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract