Mobilization of Peripheral Blood Progenitor Cells by Chemotherapy and
Granulocyte-Macrophage Colony-Stimulating Factor for Hematologic Support
After High-Dose Intensification for Breast Cancer
By Anthony D. Elias, Lois Ayash, Kenneth C. Anderson, Myla Hunt, Cathy Wheeler, Gary Schwartz, lsidore Tepler,
Rosemary Mazanet, Cathy Lynch, Stephen Pap, Jorge Pelaez, Elaine Reich, Jonathan Critchlow, George Demetri,
Judy Bibbo, Lowell Schnipper, James D. Griffin, Emil Frei 111, and Karen H. Antman
High-dose therapy with autologous marrow support results
in durable complete remissions*in selected patients with
relapsed lymphoma and leukemia &ho badhot be cured with
conventlonal dose therapy. However, substantial morbidity
and mortality result from the 3- to 6-week period of marrow
aplasia until the reinfused marrow recovers adequate hematopoietic function. Hematopoietic growth factors, particularly used after chemotherapy, can increase the number of
peripheral blood progenitor cells (PBPCs) present in systemic
circulation. The reinfusion of PBPCs with marrow has recently been reported to reduce the time t o recovery of
adequate marrow function. This study was designed t o
determinewhether granulocyte-macrophagecolony-stimulating factor (OM-CSF)-mobilized PBPCs alone (without marrow) would result in rapid and reliable hematopoietic reconstitution. Sixteen patients with metastatic breast cancer
were treated with four cycles of doxorubicin, 5-fluor0uracil,
and methotrexate (AFM induction). Patients respondingafter
the first two cycles were administered GM-CSF after the
third and fourth cycles t o recruit PBPCs for collection by two
leukapheresis per cycle. These PBPCs were reinfused as the
sole source of hematopoietic support after high doses of
cyclophosphamide, thiotepa, and carboplatin. No marrow or
hematopoietic cytokines were used after progenitor cell
reinfusion. Granulocytes EsOO/pL was observed on a median of day 14 (range, 8 t o 57). Transfusion independence of
platelets ~ 2 0 , 0 0 0pL
/ occurred on a median day of 12 (range,
8 to 134). However, three patients required the use of a
reserve marrow for slow platelet engraftment. In retrospect,
these patients were characterized by poor baseline bone
marrow cellularity and poor platelet recovery after AFM
inductlon therapy. When comparedwith 29 historical control
patients who had received the same high-dose intensification chemotherapy using autologous marrow support, time
to engraftment, antibiotic days, transfusion requirements,
and lengths of hospital stay were all significantly improved
for the patients receiving PBPCs. Thus, autologous PBPCs
can be efficiently collected during mobilization by chemotherapy and GM-CSF and are an attractive alternative t o marrow
for hematopoietic support after high-dose therapy. The enhanced speed of recovery may reduce the morbidity, mortality, and cost of high-dose treatment. Furthermore, PBPC
support may enhance the effectivenessof high-dose therapy
by facilitating multiple courses of therapy.
0 7992 by The American Society of Hematology.
DOSE
during profound myelosuppression remain the principal
causes of the morbidity and the 5% to 20% mortality of
high-dose chemotherapy. These risks are directly related to
the duration of aplasia. If hematologic reconstitution could
be enhanced, the sequelae of high-dose therapy might be
lessened and its cost reduced.
While autologous marrow is the most frequent source of
hematopoietic stem cells, circulating peripheral blood progenitor cells (PBPCs) also produce reliable and durable
hematopoietic reconstitution after high-dose therapy.7-13
The speed of recovery of the various cell lines appears
related in part to the number of nucleated cells reinfused,12J3although such correlations are hampered by the
lack of an adequate assay for human hematopoietic stem
cells. The assay generally used, granulocyte-monocyte colony-forming units (CFU-GM), measures a relatively committed hematopoietic progenitor cell. Clinical studies indicate that 15- to 50-fold more CFU-GM are required for
reliable engraftment using autologous PBPCs compared
with marrow,l*reflecting either a lower ratio of pluripotent
cells to committed progenitor cells or possibly a lack of
stromal cells reinfused with PBPCs. Seven to 12 leukaphereses are required to obtain a sufficient number of blood
mononuclear cells for engraftment, straining blood bank
and cryopreservation resources. Methods to increase
CFU-GM yields during leukapheresis have included dextran, steroids, endotoxin, and rebound after chemotherapy
(collection during recovery of the peripheral white blood
cell [WBC] c o ~ n t ) . ’ ~We
- ~ have previously shown that
administration of GM-CSF increased the number of circulating CFU-GM a median of 13-fold (2-fold to 200-fold)
INTENSIFICATIONof chemotherapeuticagents
with autologous bone marrow support (ABMT) is
currently curative in selected patients with relapsed lymphoma, leukemia, testis cancer, and neuroblastoma who
cannot be cured with conventional dose therapy.”*Laboratory evidence of a steep correlation between chemotherapy
dose and tumor response, particularly for alkylating agents,
has provided the rationale for evaluation of dose-intensive
therapy with marrow support for other, currently incurable
malignancies. Indeed, the enhanced complete response
rate and durability of complete responses in selected
patients with metastatic breast cancer is promising?
Despite marrow support, infection, and hemorrhage
From the Depamnenb of Medicine and Biostatistics, Dana-Farber
Cancer Institute; and the Division of Medical Oncology and Department of Medicine and Surgery, Beth Israel Hospital, Harvard Medical
School, Boston, MA.
Submitted September 17, 1991; accepted January 30, 1992.
Supported in part by US Public Health Service Grant No. POI CA38493 and a grant from the Maher’s Foundation. LA. and A.D.E. are
recipients of Career Development Awards from the American Cancer
society.
Address reprint requests to Anthony D. Elias, MD, Diviswn of
Clinical Oncology, Dana-Farber Cancer Institute, 44 Binney St,
Boston, IIU 02115.
The publication costs of this article were &frayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement”in accordance with 18 U.S.C. section 1734 sole& to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-4971/92/7911-0016$3.00/0
3036
Blood, Vol79, No 11 (June 11,1992: pp 3036-3044
MOBILIZED PBPC TO SUPPORT HIGH-DOSE THERAPY
3037
in untreated patients and 63-fold if administered during
hematopoietic recovery after the administration of chemotherapy.21,22
Gianni et a1 have shown that patients who received
peripheral mononuclear cells (mobilizedwith cyclophosphamide with or without GM-CSF) and marrow rapidly recovered both myeloid and platelet function after high-dose
melphalan and total body irradiatiom' This study was
designed to determine whether sufficient numbers of PBPCs mobilized with GM-CSF and chemotherapy could be
collected to provide reliable hematopoietic reconstitution
without the use of marrow.
MATERIALS AND METHODS
Eligibiliv
Patients with histologically documented metastatic breast cancer, aged 18 to physiologic 55 years, and performance status 0 to 1
were eligible. The following laboratory parameters were required:
WBC count 23,OOO/p,L, platelet count z lOO,OOO/~L,creatinine
clearance 2 60 mL/min, bilirubin and aspartate aminotransferase
(AST) ~ 1 . 5x normal, and left ventricular ejection fraction
greater than 50%. Those with tumor involving central nervous
system (CNS) or marrow were excluded, as were those with prior
pelvic radiotherapy or cumulative doxorubicin of over 240 mg/m2
(reduced to 180 mg/m2 midstudy). All patients gave written
informed consent.
grade 2 to 4 mucositis was present on day 18. For grade 4 oral
ulceration or diarrhea, 5-fluorouracil was decreased by 25%.
GM-CSF 5 pg/kg/d was administered on days 6 through 15
during cycles 3 and 4 by 24-hour intravenous infusion by Pharmacia
pump (Baxter Health Care Corp, Deerlield, IL). If grade 3 toxicity
attributable to GM-CSF occurred during cycle 3, the dose of
GM-CSF was reduced to 3 pg/kg/d for the fourth cycle. Acetaminophen was administered for GM-CSF-associated myalgias. Patients monitored temperature curves while on GM-CSF. Fever
greater than 101°F with any change in clinical status or suspected
source for infection (eg, severe mucositis) required admission for
parenteral antibiotics and supportive care. GM-CSF (Scheringl
Sandoz, Kenilworth, NJ) and carboplatin were supplied by the
National Cancer Institute (Bethesda, MD). The remaining drugs
were obtained commercially.
At least a minimal response to conventional dose doxorubicin,
5-fluorouracil, and methotrexate was required to proceed to
intensification.
BM Harvest
BM harvest was obtained after the second chemotherapy induction cycle (before exposure to GM-CSF) by standard techniques
described previously.25The reserve marrow was reinfused if less
than lOO/pL granulocytes was observed on peripheral smear on
day +28 with hypocellularity on marrow biopsy, or if at any later
time the patient failed to maintain hematopoietic function defined
as WBC less than 3,OOO/p,L and platelets less than 75,OOO/p,L.
Schema
Leukapheresisand Cyopreservation
Induction doxorubicin, 5-fluorouracil, and methotrexate (AFM)
was administered every 3 weeks for four cycles (Table 1). Each
cycle consisted of doxorubicin 25 mg/m2/d on days 3 through 5 by
bolus infusion, 5-fluorouracil 750 mg/m2/d on days 1 through 5 by
continuous infusion, and methotrexate 250 mg/m2 on day 18 with
leucovorin rescue.%Due to severe mucositis and prolonged nadirs
in the first six patients, the induction regimen was adjusted to
deliver doxorubicin by continuous infusion on days 1through 3 and
5-fluorouracil 600 mg/m2/d on days 1 through 5 by bolus infusion.
Induction therapy was held if the WBC was less than 3,OOO/pL, or
platelets less than 75,OOO/p,Lon the day of treatment. Doxorubicin
was decreased by 10% if the previous course was complicated by
documented leukopenic infection. Methotrexate was withheld if
PBPCs were collected by two 2-hour leukaphereses between
days 15 and 18 during cycles 3 and 4, provided the WBC was
greater than 1,00O/pL and increasing and platelets were greater
than 30,00O/pL. Leukaphereses were performed on a COBE
Spectra Blood Component Separator (COBE, Lakewood, CO)
using a single stage channel filter and disposable WBC blood
tubing set. Acid citrate Dextrose-Formula A (ACD-A; Baxter,
Deerfield, IL) anticoagulant, but no Ficoll or Percoll sedimenting
agents was used. As the hematocrit of the collected bum coat was
uniformly <I%, no further red blood cell (RBC) separation
procedures were performed. The buffy coat was washed with
heparin containing medium to remove fibrin and platelets that
might cause clumping during the thaw. The mononuclear cell pellet
was reconstituted in 10% dimethyl sulfoxide (DMSO) and 20%
autologous plasma in media 199 at a concentration of 2 to 5 x lo7
mononuclear cells/mL. Cryopreservation was performed using
controlled rate freezing (KRY010; Planar; TS Scientific,Perkasie,
PA) at -1°C per minute to -20°C, then -2°C per minute to
-80°C. The mononuclear cells were stored in the vapor phase of
liquid nitrogen.
Table 1. Schema
Cycle
1
Chemotherapy
AF*
GM-CSF 5 @glkgd 6-15 by continuous infusion
2-hour leukapheresis (d 15-18)
All PBPCs reinfused
EM harvest as reserve
2
3
4
5
M A F AF M A F MCTCbt
-
-
t t
t t
II
U
*Doxorubicin 25 mglmz on days 3 through 5 by bolus, 5-fluorouracil
750 mg/m2on days 1 through 5 by continuous infusion, and methotrexate 250 mg/mz on day 18 with leucovorin rescue for patients 1 through
6; ammended for patients 7 through 16 to doxorubicin on days 1
through 3 by continuous infusion, 5-fluoracil 600 mglm2 on days 1
through 5 by bolus, and methotrexate 250 mg/m2 on day 18 with
leucovorin rescue.
Kyclophosphamide(6,000 mglmz), thiotepa (500 mglm2),and carboplatin (800 mg/m2) over 4 days of continuous infusion. PBPCs were
reinfused on day 0.
IntensiJcation Therapy
Intensificationtherapy consisted of 6,000 mg/m2of cyclophosphamide, 500 mg/m2 of thiotepa, and 800 mg/m2 of carboplatin
(Cr'Cb) by 96 hours of continuous infusion. Bladder irrigation
through a 3-way Foley catheter was continued during and for 24
hours after cyclophosphamide therapy. Seventy-two hours after
completion of chemotherapy, PBPCs from the four leukaphereses
collected during induction were reinfused.
Supportive Measures
All patients received irradiated blood products (20 Gy)beginning 2 weeks before intensification. Patients were cared for in
ELIAS ET AL
3038
single rooms under reverse isolation procedures until the granulocytes were 24OO/pL on two successive determinations. Prophylactic acyclovir (400 mg orally twice per day or 250 mg/mz intravenously [IV] every 12 hours) was administered during reverse
isolation. Antiemetics included continuous infusion perphenazine
and diphenhydramine.
PBPC Reinfusion
The PBPC bags were thawed in a 37°C water bath and then
reinfused rapidly. Asymptomatic gross hemoglobinuria developed
after reinfusion in the first two patients. All subsequent patients
received hydration at 200 mL per hour for 6 hours before and for 24
hours after PBPC reinfusion. Two bags each (100 mL volume per
bag) of PBPC were infused beginning on day 0 at 4- to 6-hour
intervals until all PBPC collections were reinfused.
Laboratory Tests
Serial samples of PB for complete blood count (CBC), differential, CFU-GM colony
and percent CD34+ by fluorescenceactivated cell sorter (FACS) analysisz7were obtained on days 0,12,
15, and 17 of induction cycles 1 and 3, before each leukapheresis,
and up to 6 months after CTCb intensification. BM aspirates and
biopsies were obtained before study, at the time of marrow harvest,
at the time of completion of induction chemotherapy, at the time of
recovery of neutrophils to 500/pL, and at 6 months (or before
subsequent chemotherapy, if sooner). They were analyzed for
cellularity, differential, and colony assays (CFU-GM, burstforming unit-erythroid [BFU-E]). PB was simultaneously obtained
for CFU quantitation.
Definitions of Hematologic Response
Time to reconstitution of hematologic function was defined as
the number of days from reinfusion of stem cells (day 0) to recovery
of neutrophils to greater than 5001pL and transfusion independence for platelets ( > 2O,OOO/pL) and RBCs (hematocrit > 25%).
Hematopoietic recovery was considered durable if reconstitution
persisted to the time of last follow-up or to initiation of subsequent
therapy.
Statistical Design and Analysis
This study was designed as a pilot to investigate the feasibility of
using GM-CSF- and chemotherapy-mobilized PBPCs for reconstitution of BM function. The Wilcoxon signed rank test for paired
dataz was used to test the difference between average induction
nadirs, nadir durations, and dose intensity with and without
GM-CSF. These comparisons do not account for possible accumulating toxicity Over the four cycles. However, because the GM-CSF
cycles are the last two cycles, we feel the comparisons are biased
against the research hypotheses. The difference in induction nadir
depth and duration was compared between fast and slow engrafters to intensification with the Wilcoxon rank sum test.29
In addition, we used the immediately prior study as a historical
control group. These 29 women with stage IV breast cancer were
comparable with respect to pretreatment history and received the
identical intensification regimen, but with BM support only. The
sample size for the pilot was based on data available from the
historical control group. In this group, the time to polymorphonuclear neutrophils (PMN) greater than 500/pL appeared to follow a
lognormal distribution with a geometric mean of 20 days. Assuming
the pilot data from 15 patients was also lognormally distributed
with the same standard deviation (0.37 logs), we would be able to
detect a shift in geometric mean from 20 to 14.4 days or less with at
least 80% statistical power. Due to the presence of censored data,
the analysis of the time to engraftment of platelets and granulocytes after reinfusion was compared between patients receiving
PBPCs and those receiving BM alone with the log rank test.30
Continuous data, without censoring, such as hospital stay, units
transfused, antibiotic days, and febrile days, were compared
between patient groups with the Wilcoxon rank sum test. Dicotomous data, such as septic events, were compared with Fisher's
exact test.31Statistical tests presented are two-sided.
RESULTS
Patient Characteristics
Sixteen women entered this study between May 1989 and
April 1990. One patient, removed from study after two
cycles of induction chemotherapy due to disease progression, is therefore inevaluable for hematologic recovery and
was excluded from further analysis.
The median age was 43 years (range, 29 to 57) and the
mean performance status was 0. Antecedent hormonal
therapy had been administered to eight of nine patients
with detectable hormone receptors. Six had received prior
chest radiotherapy, nine patients had had adjuvant chemotherapy, and two had received prior chemotherapy for
metastatic disease. Six patients had had no prior chemotherapy.
Induction Phase
As shown in Table 2, the depth of granulocytopenia was
shortened by GM-CSF during cycles 3 and 4 of AFM
compared with cycles 1 and 2 (P = .002). However, duration and depth of thrombocytopenia was cumulative through
the four cycles of induction AFM with no evidence for
sparing by GM-CSF. Dose reductions of doxorubicin were
made principally for fever and neutropenia; reductions of
5-fluorouracil were mainly for mucositis. Dose delays, the
major cause of decreased dose intensity, were principally
for prolonged myelosuppression, although debility from
mucositis contributed.
Leukapheresis was performed twice between days 15 and
19 of cycles 3 and 4when the WBC increased to 2 1,000/pL
and platelets were at least 30,OOO/pL. The median mononuclear cell yield was 7.5 x 109 cells (range, 2.2 to 38.0 x lo9)
per leukapheresis. Typical leukapheresis differentials were
comparable with differentials obtained from PB smears.
The numbe'r of PBPC per kilogram collected correlated
inversely with the depth (P = .02) and duration (P = .01) of
the granulocyte nadir during the collection cycles (Fig 1).
Thus, patients with the greatest myelosuppression despite
GM-CSF had the fewest cells collected and were subsequently reinfused.
Engraftment After Intensification and Durability of
Engraftment
All evaluable patients have been followed-up for a
minimum of 12 months (range, 12 to 23) after the reinfusion
of PBPCs. No late engraftment failures have been observed.
Granulocyteengraftment. All 15 patients were evaluable
for granulocyte recovery (Table 3). The median time to
granulocyte recovery to 500/pL after PBPC reinfusion was
MOBILIZED PBPC TO SUPPORT HIGH-DOSE THERAPY
3039
Table 2. Dose Intensity and Hematopoietic Toxicity of AFM Induction Chemotherapy, and the Effect of OM-CSF on Cycles 3 and 4
Cycle of AFM
Without GM-CSF
1
With GM-CSF
2
3
4
P*
Mean days of granulocytopenia ( <500/pL granulocytes)
Range
6.0
0-18
5.3
0-15
Mean granulocyte nadir (per pL)
Range
448
0-1,650
255
0-1.080
Mean days of thrombocytopenia ( < 1OO,OOO/pL)
Range
1.8
0-7
2.3
0-8
3.1
0-17
7.6
0-20
.011
136
32-283
93
17-346
92
15-188
76
6-389
,003
81
80
83
,865
Mean platelet nadir (per pL ~ 1 0 3 )
Range
% Median dose intensityt
1.6
0-1 1
776
100-2.438
82
5.0
0-1 1
477
30- 1,692
.OM
.002
*P value comparing cycles 1 and 2 (without GM-CSF)with cycles 3 and 4 (with GM-CSF).
tMeasured by mg/m*/week normalized against the planned dose and schedule for doxorubicin and 5-fluorouracil.
14 days (range, 10 to 57). Twelve patients recovered quickly
(10 to 15 days) and three patients less quickly (26 to 57
days) (Fig 2 A). Recovery time was not related to age, prior
chemotherapy, sites of disease, or performance status, nor
was there apparent correlation between the dose of PBPCs
per kilogram and the time to granulocyte recovery. It
remains possible that a threshold phenomenon exists. The
nine patients who received greater than 4.2 x lo8mononuclear cells/kg engrafted quickly; in contrast, the speed of
recovery was slower in three of six patients who received
fewer cells. The time to engraftment appears to be inversely
correlated with the depth of the granulocytopenic nadir and
directly with the duration of granulocytopenia during induction, particularly for cycle 3 (P = .0001). Those patients
recovering quickly had less granulocytopenia during each
induction cycle than those with slower postintensification
recovery phases (Fig 3A). Response to GM-CSF also
appeared to be greater in the group ultimately engrafting
quickly.
Platelet engraftment. Fourteen patients were evaluable
for platelet engraftment. One patient died on day +26 of
10
-
9-
8
8
88
7-
8
8
8
e8
c"
8
5-
I
K
E
z
3-
8
8
8
Z
,
.
,
.
,
.
,
.
~
.
,
.
,
.
,
.
,
.
,
.
,
congestive heart failure in a clinical setting requiring
hypertransfusion of platelets. The median time to
2 20,OOO/pL and to 2 50,00O/pL platelets after reinfusion
of PBPCs was 12 days (range, 8 to 134+) and 14 days
(range, 10 to 136+), respectively. Ten patients engrafted
platelets greater than 50,OOO/kL quickly (days 10 to 22) and
four slowly (days 53 to 136f). Of these latter four patients,
one recovered by day +57. Three patients received marrow
on days +53, +134, and +136; two for persistent platelet
transfusion requirements and one to allow administration
of further therapy. One of these three patients (marked by
the arrow in Fig 2B) had recovered 2O,OOO/kL platelets by
day +13, but then developed antiplatelet antibodies; she
subsequently became platelet transfusion-dependent 4
months later and received BM on day +136. These patients
are censored at the time of marrow reinfusion for platelet
engraftment endpoints. Platelet counts in all three recovered to 25O,OOO/kL within 2 to 4 weeks of marrow
reinfusion, but not to completely normal levels. Recovery
time was not related to age, prior chemotherapy, sites of
disease, or performance status. No statistical correlation
can be made between time to engraftment of platelets and
the number of PBPC mononuclear cells per kilogram
infused; however, as was noted in granulocyte recovery, all
patients who received greater than 4.2 X lo8 cells/kg
engrafted quickly.
The 10 patients who had rapid platelet engraftment
(> 50,00O/pL) after high-dose therapy also had significantly less thrombocytopenia during AFM induction (mean,
2.7 daydcycle < lOO,OOO/~Lplatelets; range, 0 to 7.7). The
four patients with poor platelet engraftment tended to have
a longer average duration of thrombocytopenia during
AFM (mean, 7.1 daydcycle < lOO,OOO/pL platelets; range,
1.5 to 13.0) (P= .12) and greater cumulative thrombocytopenia (P= .02) (Fig 3B). Slow engraftment also correlated
with delay in treatment cycles; mean days from initiation of
induction to intensification was 128 days for the slow group
and 113 days for those with rapid engraftment (P = .011;
ELIAS ET AL
3040
Table 3. Hematopoietic Recovery After High-Dose Cyclophosphamide,Thiotepa, and Carboplatin: Sequential Trials With
Marrow or PBPC Support
BM Alone
No. of patients (responding breast cancer)
Age (range)
Treatment before intensification:
Mean no. of drugs (range)
Mean no. of regimens (range)
Received adjuvant chemotherapy
Prior chemotherapyfor metastatic disease
One inductionfor chemotherapyonly
Chest wall radiotherapy
Radiotherapyto other sites
Days from reinfusionto:
ANC > 500/pL
Platelets > 20,OOO/pL
Poor platelet engraftment (platelets
<50,00O/pL on day 50)
Toxic deaths
Documentedsepsis
Febrile days
Days on antibiotics
Days of amphotericin
Units RBCs reinfused
Units plateletstransfused
Hospital days
29
39
3.6
1.6
19
5
13
5
2
21
24
3
1
8
8
16
6
11
105
32
P Value
PBPC Alone
(27-55)
(2-6)
(1-3)
(66%)
(17%)
(45%)
(17%)
(7%)
15
43
3.7
1.6
9
2
6
6
1
(29-57)
NS
(3-5)
(1-2)
(60%)
(13%)
(40%)
(40%)
(7%)
NS
NS
NS
NS
NS
NS
NS
(12-51)
(7-83)
14
12
(10-57)
(8-134)
(sepsis)
(5)*
(2-22)
(8-103)
(0-31)
(4101)
(11-1,350)
(21-112)
4
1
2
3
10
3
8
22
24
(cardiac)
(2)'
(0-10)
(0-24)
(0-21)
(4-15)
(10-237)
(19-34)
.038
,057
NS
NS
NS
.OOM
.003
NS
,032
.001
.0006
No cytokinewas given after hematopoieticstem cell reinfusion on either trial.
Abbreviations: NS, not significant.
'5 and 2 related to central line infection, respectively.
Wilcoxon rank sum test). The patients who had poor
platelet recovery received 75% and 61% and those who had
rapid platelet recovery received 88% and 82% of the
planned dose intensities of doxorubicin (P = .01) and
5-fluorouracil (P = .12), respectively. Thus, patients who
required marrow reinfusion could be identified, in retrospect before high-dose therapy.
Marrow Cellularity and CFU-GMAssays
The median marrow cellularity before induction was 50%
(20% to 65%). All three patients who later required
marrow infusion for poor platelet engraftment had low
marrow cellularity (20% to 30%) before induction treatment. These three all had prior chemotherapy 6 to 24
months earlier, with unusually prolonged myelosuppression
noted in a single patient. All other patients had normocellular marrow. Cellularity of the marrow obtained at the time
of recovery to greater than 5001 FL PMN ranged between
10% and 80% (median, 35%).
Day 14 assays for CFU-GM and FACS analysis for
percent CD34+ cells were performed on PB serially during
cycles 1 and 3 and on leukapheresis samples from cycles 3
and 4. While a significant increase over baseline was
occasionally observed between days 12 and 17, no correlation of the number of colonies or percent CD34+ cells with
subsequent reconstitution was evident.
Hospitalization. The median hospital stay was 24 days
(range, 19 to 34). The median number of RBC and platelet
units transfused were 8 (4 to 15) and 22 (10 to 237),
respectively. The median number (range) of febrile days,
days on intravenous antibiotics, and days on Amphotericin
were 3 (0 to lo), 10 (0 to 24), and 3 (0 to 21), respectively.
Of the three patients who developed central line infections,
two had documented Staphylococcus epidemidis bacteremia. No other patients became bacteremic.
DISCUSSION
This trial shows that PBPCs recruited with chemotherapy
and GM-CSF as the sole source of hematopoietic support
provide rapid and sustained reconstitution of all cell lineages in the majority of patients. While granulocyte recovery was universal, recovery of platelet function was less
consistent. Thrombocytopenia ( < 50,000l FL), persisting to
day 50 after PBPC reinfusion, was observed in 4 of 14
patients and in three cases required reinfusion of the stored
marrow. Long-term reconstitution appeared durable in the
11 patients who recovered adequate marrow function with
PBPC alone as well as in the three who received their
back-up marrow. In retrospect, the patients requiring use of
the reserve marrow could have been identified by marrow
cellularity greater than 30% before induction therapy and
poor platelet recovery after conventional dose therapy.
The effects of PBPCs on hematopoietic reconstitution
were compared with 29 historical control patients who
received only autologous BM (Table 3). These 29 women
with metastatic breast cancer had responded to standarddose induction therapies (half had received AFM and the
other half CAF [cyclophosphamide, doxorubicin, and 5-fluorouracil]) and then received the identical intensification
regimen (CTCb) using autologous marrow support alone?*
3041
MOBILIZED PBPC TO SUPPORT HIGH-DOSE THERAPY
8
8
.
8
8
8
8
.
8
8
8
8
8
. . . . . . . . . . . . . . . . . . . . . . . .
0
10
5
A
20
15
25
30
35
40
45
50
55
80
Days followlng relnfuslon of PBPC to PYN +500/ul
10
1
i
lapped. Thus, the toxicity generated by induction chemotherapy within a particular cell lineage may imply inadequate
collection of progenitor cells within that particular line. If
this predictive model is confirmed, patients likely to recover
marrow function quickly and those who may be better
served by additional collection of PBPCs or by reinfusion of
marrow can be easily identified prospectively. Indeed, even
within the subset of six patients with less than 4.2 X @/kg
PBPCs reinfused, the three who engrafted more slowly
were those with more profound myelosuppression during
induction chemotherapy than the three who recovered
quickly.
PBPCs collected without mobilization by chemotherapy
or growth factors produce complete and permanent reconstitution of marrow function after high-dose therapy even
after potentially myeloablative regimens such as cyclophosphamide and total body irradiation.10J1.20
However, the 7 to
10 phereses required tax patient, blood bank, and cryopreservation resources. We had previously shown that
administration of GM-CSF increased the number of circulating CFU-GM a median of 13-fold (2-fold to 200-fold)
in untreated patients and 63-fold during hematopoietic
8
10
8
8
e
9 1
TT
--r
.........................
0
B
10
20
30
40
50
80
70
80
90
100 110 120 130 1 4 0
Days followlng relnfuslon of PBPC to platelets >5O,OOO/ul
Fig 2. The dose of PBPCs per kilogram does not correlate with
time to granulocyte (A) or platelet (B) recovery, but a threshold
phenomenon may exist. The arrow in (B) indicates a patient who
recovered greater than 20,0OO/pL platelets by day +13, developed
antiplatelet antibodies, and subsequently receivedBM on day 136.
+
These patients had similar pretreatment chemotherapy and
radiotherapy exposures (Table 3). The substitution of
PBPCs for marrow significantly shortened the median
number of days from reinfusion to neutrophil and platelet
engraftment by 7 days (P= .038) and 12 days (P = .057),
respectively. Median hospital stay was reduced by 8 days
(P < .001). Antibiotic requirements (P= .003) and RBC
transfusions were both significantly reduced (P= .032).
Platelet transfusion requirements were markedly reduced
(P= .001). Failure of platelet engraftment has been observed in 9% to 23% of patients when using autologous
marrow as s ~ p p o r t ~and
~ ,was
~ ~observed
~ , ~ ~ in one of 29
breast cancer patients using CTCb with
The adequacy of specific cell lineage progenitor collection and subsequent engraftment could be predicted by
specific cell-lineage toxicity caused by conventional-dose
induction chemotherapy. For example, patients with slower
granulocyte recovery after high-dose cyclophosphamide,
thiotepa, and carboplatin had protracted, profound granulocytopenia during all four conventional-dose AFM induction cycles. Parallel findings of prolonged thrombocytopenia during conventional induction chemotherapy were
observed for patients with delayed platelet recovery after
high-dose therapy. These two groups only partially over-
B
CM-CSF
Platelet Enlpafl
(-1
(+)
Fast (ado)
(-)
(+)
slow (n=4)
Fig 3. OM-CSF shortens granulocytopenia (A), but does not prevent cumulative thrombocytopenia (B). The time to engraftment of
the particular cell lineage correlates directly with the durations of
granulocytopenia (A) and thrombocytopenia (B) during induction. I-),
Cycles 1 and 2 without GM-CSF; I+), cycles 3 and 4 with GM-CSF. The
patients who are "fast" and "slow" in (A) and (B) are defined by the
patient distributionshown in Fig 2A and B, respectively.
ELlAS ET AL
3042
recovery after the administration of chemotherapy.21s22In
combination with autologous marrow, cytokine- or chemotherapy-mobilized PBPCs also enhanced hematopoietic
r e c o ~ e r y . ~Gianni
,~~,~
et~a1 determined that marrow plus
PBPCs from 2 to 3 leukaphereses during recovery after 7
g/m2 of cyclophosphamide (with and without GM-CSF)
produced early reconstitution of both myeloid and platelet
function after high-dose melphalan and total body irradiation.= The number of CFU-GM per kilogram reinfused
correlated well with the time to absolute neutrophil count
(ANC) recovery. Peters et a1 have combined GM-CSFmobilized PBPCs and marrow after high-dose cyclophosphamide, carmustine, and ~isplatin.3~
GM-CSF alone increased
circulating CFU-GM 10-fold and CD34+ (a cell surface
antigen carried by the progenitor cell population) cells up
to 11% of the leukapheresed mononuclear cell fraction.
These patients experienced fewer days of absolute granulocytopenia (ANC < lOO/pL) compared with controls who
received marrow alone followed by GM-CSF.35 G-CSF also
appears to mobilize CFU-GM into the peripheral circulation. Investigators in Melbourne have observed more rapid
ANC and platelet recovery in patients receiving both
PBPCs collected after G-CSF and autologous marrow.37
The mechanisms by which hematopoietic cytokines increase circulating PBPCs are not well understood. They
may provide a proliferative stimulus to expand the population of progenitor cells within the marrow and PB compartments, a differentiation stimulus to increase circulating
progenitor cells (at the expense of stem cells responsible for
self-renewal), or an alteration in adhesion molecules on the
cell membrane that ordinarily regulate the release of cells
from the marrow into the circulating c ~ m p a r t m e n t ? The
~.~~
fact that G-CSF-stimulated PBPCs together with marrow
appears to enhance platelet engraftment37 despite its lack
of stimulation of thrombopoiesis suggests that the release
of progenitor cells by alterations in adhesion molecules may
be at least partly the explanation. Mobilization by chemotherapeutic agents may be due to high marrow cytokine
concentrations in response to relative aplasia and may not
be specific for a given chemotherapy agent itself.
The timing of GM-CSF within the cycle of induction
chemotherapy may be important to optimize collection of
mobilized PBPC. Gianni et a1 showed that GM-CSF is more
effective in ameliorating myelosuppression due to cyclophosphamide when begun on day +1 than on day +5.40 In a
companion study of the same patients$l Siena et a1 reported that the number of CD34+ cells per kilogram (in
particular, the CD34+/CD33+ subset; a phenotype describing more mature progenitor cells) correlated with the time
to early hematopoietic recovery.42The presence of these
committed progenitor cells correlated strongly with the
growth of day 14 CFU-GM. The rebound of CD34+ cells
and CFU-GM was observed when GM-CSF was begun on
day +1, but not if delayed to day +5 of the cycle." These
studies suggest that mobilization of PBPCs and enhanced
maturation by GM-CSF optimally requires exposure over a
period of days. In our study, we were unable to document a
consistent rebound effect on CD34+ cells in the circulation
after cycles 3 and 4, perhaps because GM-CSF was begun
on day +6 of the cycle. The intrapatient variation of
CFU-GM colonies rendered this assay problematic for
analysis. Despite the lack of a rebound effect on CD34 cells,
early reconstitution was observed clinically.
Rapid engraftment possible with PBPC support should
enhance the evaluation of dose-intensive therapy. Early
reconstitution should reduce serious infectious and bleeding complications, antibiotic courses, hospital stays, and
fiscal costs. Despite the four phereses to collect PBPCs per
patient, the use of pheresis resources in the blood bank
actually decreased because of these patients' dramatically
reduced requirements for platelet support. Decreased risks
and cost should permit the study of dose-intensive therapy
in earlier stage poor prognosis diseases.
On a more fundamental level, PBPCs may allow for the
safe administration of sequential high-dose regimens. Single courses of dose-intensive therapy currently cure only a
minority of patients. Curative conventional-dose chemotherapy has been successful in situations in which active drugs
are used in combination for multiple courses. Multiple
cycles of high-dose chemotherapy have been toxic, expensive, and impractical. A preliminary report by Shea et a1
indicates that PBPCs and GM-CSF adequately supported
repeated cycles of 1,200 mg/m2 of carboplatin, doses that
could not be repeated for three cycles due to profound
thrombocytopenia when using GM-CSF alone.44 Thus,
testing the concept of dose-rate becomes feasible. On a
cautionary note, these trials must consider the possibility
that PBPCs may contain enough mature progenitors to
support patients through acute myelosuppression of multiple high-dose courses, but might not be sufficient to prevent
cumulative myeloablative effe~ts.4~
Unanswered questions regarding the optimal technique
for collection of PBPCs remain. Optimal scheduling of the
growth factor with respect to the chemotherapy may enhance the number of circulating PBPCs and the efficiency
of collection. Other cytokines andlor combinations may
optimize mobilization. In the laboratory, interleukin-3 increases circulating CFU-GM and is synergistic in this effect
with GM-CSF.46-48Preclinical modelling may also guide
determination of optimal chemotherapy agents for inducing a rebound in circulating CFU-GM or CD34+ cells and
provide an understanding of the regulatory mechanisms of
progenitor cell traffic between the BM and PB compartments.
ACKNOWLEDGMENT
We thank the house staff of the Beth Israel Hospital and the
Brigham and Women's Hospital and the nursing staffs of IZW at
the Dana-Farber Cancer Institute and 4s of the Beth Israel
Hospital. Sheila Daigle provided excellent data management.
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