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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 864-869
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Successful in vivo purging of CD34-containing peripheral blood
harvests in mantle cell and indolent lymphoma: evidence for a role
of both chemotherapy and rituximab infusion
Michele Magni,
Massimo Di Nicola,
Liliana Devizzi,
Paola Matteucci,
Fabrizio Lombardi,
Lorenza Gandola,
Fernando Ravagnani,
Roberto Giardini,
Giuseppe Dastoli,
Corrado Tarella,
Alessandro Pileri,
Gianni Bonadonna, and
Alessandro M. Gianni
From the Department of Oncology, University of Milan, Milan, Italy;
the Divisions of Medical Oncology C, Radiotherapy, and Pathology,
National Cancer Institute, Milan, Italy; the Department of Hematology,
University of Turin, Turin, Italy; and Roche SpA, Milan, Italy.
 |
Abstract |
Elimination of tumor cells ("purging") from hematopoietic stem
cell products is a major goal of bone marrow-supported high-dose cancer chemotherapy. We developed an in vivo purging method capable of
providing tumor-free stem cell products from most patients with mantle
cell or follicular lymphoma and bone marrow involvement. In a
prospective study, 15 patients with CD20+ mantle cell or
follicular lymphoma, bone marrow involvement, and polymerase chain
reaction (PCR)-detectable molecular rearrangement received 2 cycles of
intensive chemotherapy, each of which was followed by infusion of a
growth factor and 2 doses of the anti-CD20 monoclonal antibody
rituximab. The role of rituximab was established by comparison with 10 control patients prospectively treated with an identical chemotherapy
regimen but no rituximab. The CD34+ cells harvested from
the patients who received both chemotherapy and rituximab were
PCR-negative in 93% of cases (versus 40% of controls;
P = .007). Aside from providing PCR-negative harvests, the
chemoimmunotherapy treatment produced complete clinical and molecular
remission in all 14 evaluable patients, including all 6 with mantle
cell lymphoma (versus 70% of controls). In vivo purging of
hematopoietic progenitor cells can be successfully accomplished in most
patients with CD20+ lymphoma, including mantle cell
lymphoma. The results depended on the activity of both chemotherapy and
rituximab infusion and provide the proof of principle that in vivo
purging is feasible and possibly superior to currently available ex
vivo techniques. The high short-term complete-response rate observed
suggests the presence of a more-than-additive antilymphoma effect of
the chemoimmunotherapy combination used.
(Blood. 2000;96:864-869)
© 2000 by The American Society of Hematology.
| |
Introduction |
Myeloablative therapy followed by autologous
hematopoietic progenitor cell rescue has an established, albeit not
clearly defined, role in the management of non-Hodgkin lymphoma
(NHL).1,2 One major obstacle to autologous stem cell
transplantation in NHL is bone marrow and peripheral blood cell
involvement with malignant cells. In fact, even if relapses after
autologous transplantation usually result from residual cancer in the
patient, cancer cells in bone marrow grafts contribute to relapse, as
was demonstrated by gene-marking studies in patients with acute and
chronic leukemia3,4 and patients with
neuroblastoma.5 A role for bone marrow purging in
autologous bone marrow transplantation for patients with NHL was
suggested by studies that strongly indicated that residual lymphoma
cells contribute to relapse.6 Whether these data also apply
to peripheral blood cell grafts is unknown, although the presence of
contaminating tumor cells in a product that is to be reinfused is of
obvious concern.
With the aim of eliminating malignant cells from the graft, various ex
vivo techniques involving the use of monoclonal antibodies or
chemotherapeutic drugs have been developed.7 While
generally effective, these purging techniques are labor intensive,
delay hematopoietic recovery after transplantation, and substantially increase the costs of treatment.8
It was shown that hematopoietic progenitor cells harvested from the
peripheral blood after high-dose conventional chemotherapy in patients
with indolent lymphomas were tumor-free in a substantial proportion of
cases9 and that in vivo administration of the monoclonal
anti-CD20 antibody rituximab caused a rapid depletion of peripheral
blood B cells in both cynomolgus monkeys10 and patients.11 This evidence was subsequently confirmed by the observation that when rituximab is given in combination with
standard-dose chemotherapy, conversion of bone marrow and peripheral
blood cells to negativity for tumor cells on polymerase chain reaction
(PCR) assessment frequently occurs.12
In a study based on these findings, we investigated the ability of
rituximab, given after 1 or 2 cycles of nonmyeloablative high-dose
chemotherapy, to allow harvesting of peripheral blood progenitor cells
free of contaminating lymphoma cells (in vivo purging) in 15 patients
with either relapsed or refractory indolent lymphoma or mantle cell
lymphoma (MCL). For each enrolled patient, we studied the extent of the
mobilization and procurement of CD34+ cells, the presence
of PCR-detectable lymphoma cells in the harvested product, and the
kinetics of hematopoietic engraftment after reinfusion of the in vivo
purged cells in patients who had myeloablation. The precise role of the
antibody was assessed by comparison with results in a cohort of 10 consecutively seen similar patients who were treated with the same
high-dose chemotherapy regimen but not rituximab.
| |
Patients and methods |
Patients
Between December 1996 and March 1999, 25 consecutively seen patients
with either untreated mantle cell lymphoma or refractory or relapsed
indolent NHL received high-dose sequential (HDS) chemotherapy with
autologous hematopoietic progenitor cell support. Eligibility criteria
included written informed consent; age, 60 years or younger; absence of
severe organ dysfunction not due to tumor; no previous viral infections
(hepatitis B or C or human immunodeficiency virus); a histologically
confirmed diagnosis of mantle cell lymphoma or of indolent lymphoma,
either refractory to or relapsed within 1 year after first-line
polychemotherapy and requiring treatment; expression of CD20 by
lymphoma cells; and availability of a molecular probe for PCR
amplification of DNA to assess minimal residual disease (MRD).
Characteristics of patients given HDS alone and patients given
rituximab with HDS (R-HDS) are shown in Table
1. The study included 3 consecutive cohorts
of patients whose treatment was dictated exclusively by the
availability of rituximab. Thus, the first 10 and the last 5 enrolled
patients received R-HDS, whereas the 10 patients seen consecutively
between those groups (that is, enrolled after completion of the initial
pilot study in 10 patients and before the antibody became commercially
available) served as controls (HDS without rituximab).
Treatment plan
All patients who had not previously received doxorubicin were
initially treated with 2 to 3 courses of this agent (75 mg/m2 of body-surface area given intravenously on day 1),
prednisone (40 mg/m2 orally given from day 1 to day 21),
and vincristine (1.4 mg/m2 given intravenously on day 1).
Patients with refractory or relapsed disease who had received
doxorubicin previously and patients with little or no response
to the initial course described above received 2 to 3 cycles of
cisplatin-high-dose cytarabine-dexamethasone chemotherapy.13 After the initial
standard-dose phase, all patients received the 4-step high-dose
sequence shown in Figure 1, including intravenous administration of high-dose cyclophosphamide (7 g/m2),14 high-dose cytarabine (either 1.5 or 2 g/m2 every 12 hours for 6 consecutive days),15
high-dose melphalan (180 mg/m2),16 and
high-dose mitoxantrone plus melphalan (60 and 180 mg/m2,
respectively).17 The protocol entailed high-dose drug
administrations every 3 weeks, depending on hematologic and
nonhematologic toxicity. Patients with lesions larger than 5 cm at
study entry or with documented or suspected residual disease after the
end of treatment received consolidation radiotherapy according to
procedures described previously.18
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| Fig 1.
Overall treatment plan, including both the in vivo
purging phase (cyclophosphamide and cytarabine) and subsequent
myeloablative antilymphoma therapy (melphalan and mitoxantrone plus
melphalan).
The doses were as follows: cyclophosphamide, 7 g/m2 of
body-surface area; cytarabine, 1.5 to 2 g/m2 every 12 hours
for 6 consecutive days; recombinant granulocyte colony-stimulating
factor (CSF) or recombinant granulocyte-macrophage CSF, 5 µg/kg of
body weight/day; reinfusion 1, 2 × 106
CD34+ cells/kg, irrespective of polymerase chain reaction
(PCR) status; and reinfusions 2 and 3, 5 × 106 and
at least 8 × 106 CD34+ cells/kg,
respectively, only if PCR negative. Intervals between cycles are
approximate.
|
|
Rituximab (Roche, Milan, Italy) was diluted and administered as an
intravenous infusion according to the manufacturer's instructions. Patients in the R-HDS group received 6 overall infusions of the antibody (375 mg/m2). Rituximab infusions 1 and 2 were
administered 48 hours after cyclophosphamide and 24 hours before the
first leukapheresis, respectively. Rituximab infusions 3 and 4 were
given 24 hours after the last dose of cytarabine and 24 hours before
the first planned leukapheresis after administration of cytarabine,
respectively. Rituximab infusions 5 and 6 were given approximately 28 and 35 days, respectively, after the final stem cell autograft, after administration of mitoxantrone and melphalan.
The supportive care given during the entire course of HDS therapy
(including administration of a hematopoietic growth factor) was
described in detail previously.18,19 To hasten
hematopoietic recovery and reduce hematologic toxicity, each patient
received 3 progenitor/stem cell reinfusions, both after the
nonmyeloablative course of high-dose cytarabine (reinfusion 1) and
after the 2 subsequent submyeloablative/myeloablative courses of
melphalan and mitoxantrone plus melphalan (reinfusions 2 and 3, respectively). The minimum target doses of CD34+ cells per
kg of body weight to be reinfused after each of the 3 autografts were
2 × 106, 3 × 106, and
5 × 106, respectively. The small amount of
CD34+ cells to be reinfused after cytarabine treatment was
allowed to contain PCR-detectable disease, whereas autografting of
products free of residual lymphoma was mandatory after administration
of melphalan (reinfusion 2) and mitoxantrone plus melphalan (reinfusion 3). Each patient underwent the minimum amount of peripheral blood harvesting necessary to achieve this goal.
The timing and number of collections were prospectively guided by
real-time assessment of circulating progenitor counts,20 as
well as by results of overnight PCR analysis on a sample of leukapheresed cells. If the first harvest after cyclophosphamide was
both quantitatively adequate for all 3 reinfusions (ie, it yielded  10 × 106 CD34+ cells/kg) and PCR
negative, no additional leukaphereses were performed. If these criteria
were not met , a second harvest was attempted during the
rapid recovery phase that followed high-dose cytarabine therapy, and
the cells were stored overnight at 4°C until the results of PCR
analysis were available. If the cells were still PCR positive, the
harvested cells were incubated with anti-CD19 monoclonal antibody and
subjected to in vitro negative selection using a Miltenyi Super MACS
cell-sorting system.21 The overall purging procedure,
including (if required) ex vivo purging, was considered successful when
it was possible to harvest a minimum of 8 × 106
PCR-negative CD34+ cells/kg, ie, the total amount required
for reinfusions 2 and 3.
Monitoring, harvesting, and processing of hematopoietic progenitor
cells
Hematopoietic progenitor cells in peripheral blood and leukapheresis
components were assessed by counting total CD34+
cells by direct immunofluorescence flow cytometry with the
phycoerythrin-conjugated HPCA-2 CD34 antibody (Becton Dickinson, San
Jose, CA) as previously described.21
Leukaphereses were performed during growth-factor expanded mobilization
of progenitor cells occurring after administration of either high-dose
cyclophosphamide, high-dose cytarabine, or both. On the first recovery
day after chemotherapy that the CD34+ cell count reached at
least 0.02 × 109/L, the patients
received 1 dose of rituximab; 24 hours later, they underwent
leukapheresis procedures until the target number of CD34+
cells was collected. Mononuclear cells were collected with use of a
novel automated leukapheresis system (Auto PBSC Spectra; COBE,
Lakewood, CO), as previously described.22
Molecular monitoring of MRD was accomplished by assessing DNA samples
from peripheral blood, bone marrow, and leukapheresis products with use
of either nested PCR amplification of either the bcl-2/IgH or bcl-1/IgH
translocation or semi-nested amplification of clonal rearrangement of
IgH genes, essentially as described by Corradini et al9 and
Andersen et al.23 The limit of detection of MRD was
reproducible at the level of 10 6 for the bcl-2/IgH
translocation and at 105 for the other 2 rearrangements. The progenitor cells to be autografted were processed,
cryopreserved, thawed, and reinfused as described previously.24
Response to treatment and statistical analysis
Complete response to treatment was defined according to
international working group recommendations, which entail complete disappearance of all detectable clinical and radiographic evidence of
disease, regression of lymph nodes and any enlarged organ to normal
size, and clearance of lymphoma involvement in the bone marrow on
morphologic analysis.25
The characteristics of the patients and their response to treatment
were compared with the Fisher exact test. The significance of
differences among the different groups in myelosuppression duration,
yield of CD34+ cells, number of leukaphereses performed,
and transfusions required was calculated with the nonparametric
Mann-Whitney U test or t test, as appropriate.
| |
Results |
Patient characteristics
The characteristics of the 25 patients enrolled into the study are
listed in Table 1. All 15 patients with follicular or marginal-zone
lymphoma had previously received 1 line of polychemotherapy (either
cyclophosphamide, hydroxydaunomycin, vincristine, and prednisone
[CHOP]; cyclophosphamide, vincristine, and prednisone; or
fludarabine, mitoxantrone, and dexamethasone), with or without local-regional radiotherapy. At the time of entry into the study, these
patients had progression of disease requiring treatment that occurred
either after an initial partial response (12 patients) or a complete
response that lasted less than 1 year (3 patients). The bcl-2/IgH
rearrangement was detected in 10 of the 14 patients with follicular
lymphoma. All 15 patients in this subgroup had PCR-detectable disease
in the bone marrow; in 10, histologic studies showed marrow involvement.
The diagnosis of MCL was confirmed in all 10 enrolled cases by
expression of CD5 and lack of expression of CD23 on the tumor cells. No
patient with MCL had previously received chemotherapy or radiotherapy.
The bcl-1/IgH rearrangement was detected in 6 patients and absence of
the bcl-2/IgH rearrangement in all 10. All MCL patients had involvement
of multiple nodal sites and histologic evidence of bone marrow
involvement, which was extensive in 7. The latter patients had also
circulating lymphoma cells in the peripheral blood, confirmed by
immunophenotyping (ie, CD20+, CD5+, and
CD23 cells), in amounts ranging from
0.04 × 109/L to more than
1.0 × 109/L (in 4 patients).
Table 1 shows that the patient characteristics that might have
influenced the purging (ie, disease extent and bone marrow and
peripheral blood involvement by lymphoma cells) were equally represented in the 2 groups. The major differences between the groups
were a higher number of men, MCL diagnoses, and molecular diseases (IgH
rearrangements) in the R-HDS group. All these differences most likely
reflect a single factor, ie, a chance overrepresentation of mantle cell
histologic features in the group that received rituximab. In fact, MCL
occurs predominantly in men,26 and because of considerable
heterogeneity in the site of the underlying molecular rearrangement,
the bcl-1/IgH translocation can be amplified by PCR in less than half
of MCLs.23
Mobilization and harvesting of CD34+ cells after
cyclophosphamide and cytarabine
Table 2 shows the peak values of
CD34+ cells circulating in the peripheral blood during the
growth-factor-supported recovery phase, with and without rituximab
infusion. No significant differences were observed among the 4 groups
analyzed, indicating that rituximab infusion had no adverse effect on
mobilization and that previous cyclophosphamide treatment did not
impair progenitor mobilization after cytarabine administration, which
was done soon afterward. This surprising lack of negative
influence27 was confirmed by statistical analysis with a
paired test of peak values in the same patient. Likewise, no
significant differences were observed among the 4 groups in the day of
the first apheresis, the number of aphereses, and the total number of
CD34+ cells harvested (Table 2).
PCR analysis of bone marrow, peripheral blood, and leukapheresis
products
Major and significant differences were observed in the ability to
harvest PCR-negative CD34+ cells. As shown in Table 2 and
Figure 2, after administration of
cyclophosphamide, PCR-negative products were harvested in 2 patients
(20%) who did not receive rituximab but in 5 patients (33%) who did.
On the other hand, after administration of cytarabine, the proportion
of harvests free of lymphoma cells increased to 40% in the HDS group
and to 93% in the R-HDS group (P = .007).
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| Fig 2.
Cumulative proportion of patients from whom PCR-negative
harvests were obtained after either cyclophosphamide (p-CTX),
cytarabine (p-AraC), or cytarabine plus ex vivo purging (ex vivo
purging).
The latter group includes only patients in the high-dose sequential
therapy arm.
|
|
Ex vivo purging was planned for all 7 patients in whom in vivo purging
had failed (6 patients in the HDS group and 1 in the R-HDS group), but
it was actually performed in 5. The other 2 patients did not undergo ex
vivo purging because of lack of an adequate number of CD34+
cells (patient 10) and mechanical failure of the cryopreservation device (patient 20).
Thus, the rate of success in harvesting PCR-negative products remained
higher in the R-HDS group, although not significantly so, even after ex
vivo purging (93% versus 80%; Figure 2). This qualitative advantage
was strengthened by quantitative differences in the overall yield of
CD34+/PCR-negative cells between the 2 groups (Figure
3). In fact, when we used an
intent-to-treat analysis to compare the ability of the 2 different
procedures to yield lymphoma-free progenitors, the R-HDS regimen was
clearly found to be superior (mean yield of
CD34+/PCR-negative cells per kg, 26 versus 13;
P = .029).
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| Fig 3.
Total yield of PCR-negative and CD34+ cells
harvested from the peripheral blood.
Each patient underwent the minimum number of procedures sufficient to
complete the therapeutic program (Figure 1). Boxes extend from the 25th
to the 75th percentile, with the horizontal line indicating the median.
Whiskers extend from the largest to smallest values.
|
|
The in vivo purging efficacy of the combination of high-dose
chemotherapy and rituximab was further emphasized by a subgroup analysis of patients with MCL, a disease well known to be recalcitrant to both in vivo9 and in vitro purging.23 In
fact, in vivo purging was successful in all 7 MCL patients in the R-HDS
group. Of note, 4 of these 7 patients (patients 18, 19, 21, and 22) had initial massive bone marrow involvement and leukemic findings in the
peripheral blood (basal CD5+/CD23
mononuclear cells counts of 0.18, 1.7, 3.8, and
6.9 × 109/L , respectively).
Informative data were derived from simultaneous PCR analyses of bone
marrow, peripheral blood, and leukapheresed cells done both after
administration of cyclophosphamide and after administration of
cytarabine. If the bone marrow was PCR positive, leukapheresis products
were invariably PCR positive in the HDS group (12 of 12 cases).
Interestingly, in the R-HDS group, 4 of 13 leukaphereses done in
patients with PCR-positive bone marrow were PCR negative. A lack of
absolute concordance between the molecular status of bone marrow and
leukapheresis products also emerged from analysis of patients with
PCR-negative bone marrow. In fact, of 24 total leukaphereses performed
in patients with PCR-negative bone marrow, 4 showed a PCR-positive
signal (3 in the HDS group and 1 in the R-HDS group). Similarly low was
the concordance between peripheral blood and leukapheresis
products. In fact, although all 18 patients with PCR-positive blood had
PCR-positive aphereses, the reverse was not true: 8 of 18 harvests from
patients with PCR-negative peripheral blood were contaminated with
lymphoma cells. Thus, the molecular status of neither bone marrow nor
peripheral blood could be used as a convenient surrogate test for
predicting tumor-free leukaphereses and prospectively guiding
harvesting procedures.
Hematopoietic recovery after autografting
Rituximab treatment had no adverse effect on the capacity of in vivo
purged cells to support a rapid and complete hematopoietic reconstitution after the final myeloablative course (mitoxantrone and
melphalan). In fact, no significant difference was observed between the
HDS and R-HDS groups in the mean number of days spent with less than
0.1 ×109 neutrophils/L (5.5 versus 6.2 days) and
less than 0.5 ×109 neutrophils/L (7.4 versus 7.7 days), whereas a borderline or significantly favorable
effect was observed for platelet recovery. The patients who received
autografts of cells exposed in vivo to rituximab had a mean of 7 days
with a platelet count below 25 × 109/L
(versus 13.6 days in the HDS patients; P = .59)
and required significantly fewer single-donor platelet transfusions
(mean, 5 versus 10; P = .05). They also required fewer
transfusions of red blood cells (mean, 3.6 versus 9;
P = .03). Both beneficial effects most likely reflected the
higher dose of CD34+ cells autografted in the R-HDS
patients (Figure 3).
Overall treatment toxicity and tumor response
There were 2 treatment-related deaths, 1 in each group.
Both occurred after the second autotransplantation. One
patient with a history of cardiac arrhythmia died suddenly on day 14 after mitoxantrone and melphalan administration after an
otherwise uneventful recovery, and 1 patient positive for antibody to
hepatitis B core antigen (HBcAb) and negative for
HBsAg died of fulminant hepatitis B on day 180 after administration of mitoxantrone and melphalan.
The 15 patients enrolled in the study received a total of 88 infusions
of rituximab (375 mg/m2). Only 2 patients (2.3%) had
adverse events with infusion. Both events development of a rash in 1 patient and dyspnea in the otheroccurred during the first infusion
only and were rated as being of grade 1 severity.
Of the 10 evaluable patients who received HDS, 1 patient died
of fulminant hepatitis B, 2 additional patients did not achieve complete remission and died of disease progression, and 1 patient never
attained molecular eradication of the disease in the bone marrow. After
a median follow-up duration of 10 months, 7 patients in the HDS group
were free of both clinical and molecular disease. Of the 14 R-HDS
patients evaluable for response (1 died as a result of toxicity too
early for an accurate assessment), all achieved a complete clinical and
molecular response and all were relapse-free after a median
follow-up duration of 14 months. Of note, all 9 evaluable patients with
MCL achieved a complete and durable response.
| |
Discussion |
Contamination of bone marrow collections by tumor cells can
contribute to relapse. This fact, proved formally by gene-marking studies,3-5 represents the strongest argument for using
measures to eliminate malignant cells from the graft (purging). In
fact, although it will be difficult to proof definitively that purging has a role in improving disease-free survival, it is hard to justify immediately following the delivery of a myeloablative therapy course
aimed at eradicating disease with an intravenous infusion of tumor
cells capable of causing relapse.
The ex vivo techniques for purging, while usually effective in reducing
by several logs the amount of contaminating tumor cells, have severe
limitations. In fact, these techniques are labor intensive, they may
delay bone marrow recovery as a consequence of substantial loss and
variable damage of stem and committed progenitor cells, and they
increase costs. Furthermore, elimination of accessory and lymphoid
cells by techniques for selecting stem cells might adversely influence
immune reconstitution or antitumor effects. A variety of strategies
have been proposed as alternatives to ex vivo purging. Collectively
referred to as "in vivo purging," these methods are aimed at
reducing or eliminating contaminating cells by means of treatments
delivered to or procedures carried out in patients. The techniques
include harvesting of peripheral blood instead of marrow cells (a
so-far largely unproved method) and administration of cytotoxic
drugs9,28-30 or monoclonal antibodies before the
harvest.12,31 However, in many cases, successful in vivo
purging has only been inferred from indirect clinical data,29-31 whereas in others, residual neoplastic cells
were detected by immunophenotypic or cytogenetic methods with
relatively low sensitivity and specificity. Indeed, when Boqué
and coworkers32 used reverse transcriptase-PCR to analyze
hematopoietic progenitors harvested from patients with chronic myeloid
leukemia according to the program developed by Carella et
al,28 all Philadelphia chromosome-negative bags were
positive for the bcr/abl translocation.
Proof that an intensive in vivo chemotherapy treatment can foster the
subsequent collection of lymphoma-free progenitors was provided by
Corradini et al,9 who harvested PCR-negative peripheral blood products from 1 of 9 patients (12%) with mantle cell lymphoma and 8 of 19 (42%) with follicular lymphoma. Furthermore, toxicologic studies in cynomolgus monkeys10 showed that infusion of the monoclonal antibody rituximab caused a rapid and reproducible clearance
of CD20+ cells from the blood of treated animals. These 2 observations provided the rationale for the current study. In fact,
only subsequently12 was it reported that a combination of
rituximab and CHOP chemotherapy converted bcl-2/IgH-positive marrow to
PCR negativity in a subset of patients with low-grade B-cell lymphoma,
a finding that provides additional independent support for our in vivo
purging strategy.
The present study demonstrated that in vivo purging can be accomplished
successfully in most CD20+ mantle cell and follicular
lymphomas (93%) and that successful purging resulted from both
chemotherapy and immunotherapy. The role of chemotherapy was confirmed
by the facts that chemotherapy alone was able to provide PCR-negative
harvests in 40% of cases (Figure 2) and that the proportion of
PCR-negative products was invariably higher when harvesting was
carried out after the second course of chemotherapy (ie, high-dose
cytarabine) (Figure 2 and Table 2). These results are in agreement with
those reported by Corradini et al.9
The important role of rituximab was demonstrated by the fact that
PCR-negative harvests were obtained from 93% of the patients who had
received 4 rituximab infusions but only 40% of the controls (P = .007; Figure 2). A possible bias in our assessment of
the in vivo purging role of rituximab was the higher proportion of patients in the R-HDS group whose MRD was examined by PCR amplification of the less sensitive IgH rearrangement. This unbalance reflects the
presence of a higher number of patients in the R-HDS group than in the
HDS (8 [53%] versus 3 [33%]) with histotypes for which a
bcl-2/IgH probe was not applicable (7 cases of MCL and 1 case of
mucosa-associated lymphoid tissue lymphoma). The in vivo purging role
of rituximab in this small population could not be determined by subset
analysis but is strongly suggested by comparison with results obtained
in studies using the same technique adopted here to detect MRD in bone
marrow23 and peripheral blood harvests9,33 from
patients with MCL. In fact, all 7 patients in the R-HDS group had
PCR-negative harvests. For comparison, in the study by Corradini et
al,9 high-dose chemotherapy alone was effective in 1 of 9 cases, whereas immunologic ex vivo purging failed to eradicate PCR-detectable disease in 15 of the 17 MCL patients reported on by
Andersen et al23 and all 3 MCL patients treated by Tarella et al.33
The addition of ex vivo purging converted to PCR negativity the
harvests from 5 of the 6 HDS patients in whom in vivo purging had
failed (Table 2). This increased the total success rate in the HDS
group to 80%, close to the 93% rate in the R-HDS arm (P not
significant). However, the total yield of CD34+ cells was
significantly lower in the HDS group (P = .029; Figure 3), a
finding that could explain the delayed marrow recovery observed in
those patients. In fact, recovery after transplantation, especially platelet recovery, is directly correlated with CD34+ cell
dose.8
Of note, although administration of high-dose cytarabine occurred close
to administration of high-dose cyclophosphamide, it did not influence
the extent of CD34+ cell mobilization (Table 2). This
effect was surprising and in contrast with our previous finding that
short intervals between high-dose drug courses severely impair
progenitor mobilization.27 However, a different sequence of
drugs was used here (ie, cyclophosphamide followed by cytarabine
compared with etoposide followed by cyclophosphamide in our previous
study27), and recovery after cytarabine was hastened by
reinfusion of progenitor cells.
Finally, in assessing overall efficacy, it is tempting to speculate
that the very high complete response rate observed in the R-HDS group
might reflect an important therapeutic role of rituximab given during
maximal expansion and activation of the granulocyte-macrophage system
by concomitant treatment with growth factors.34
| |
Acknowledgments |
We thank Dr Marco Bregni, Dr Salvatore Siena, and the staff at the
various INT Units for expert patient care; Marco Milanesi and Paolo
Longoni for technical assistance; and Dr Luca Baldini, Dr Attilio
Gabbas, Dr Giovanni Martinelli, Dr Enrico Pogliani, and Dr Carlo
Tondini for referring patients with MCL.
| |
Footnotes |
Submitted November 18, 1999; accepted March 29, 2000.
Supported in part by AIRC and by Ministero della Sanità
grant ICS 030.1/RF96.278.
M.M. and M.D.N. contributed equally to this work.
Reprints: Alessandro M. Gianni, Cattedra di Oncologia Medica,
Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy; e-mail:
alessandro.gianni{at}unimi.it.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction